Migrastatin analog compositions and uses thereof

ABSTRACT

In one aspect, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of a compound of general formula (I), 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1 -R 6 , R a -R c , Q, Y 1 , Y 2  and n are as defined herein, whereby the composition is formulated for administration to a subject at a dosage between about 0.1 mg/kg to about 50 mg/kg of body weight. 
           
         
       
    
     In another aspect, the present invention provides a method for treating breast tumor metastasis in a subject comprising administering to a subject in need thereof a therapeutically effective amount of the inventive composition described directly above and a pharmaceutically acceptable carrier, adjuvant or vehicle.

PRIORITY CLAIM

The present application is a divisional of U.S. patent application Ser.No. 10/551,152, filed Sep. 25, 2006 now abandoned, which is a U.S.national phase application under 35 U.S.C. §371 of InternationalApplication No. PCT/US04/09380, filed Mar. 26, 2004, which claimspriority to U.S. Provisional Application Nos.: 60/458,827, filed Mar.28, 2003, and 60/496,165, filed Aug. 19, 2003; the entire contents ofeach of the above-referenced applications are hereby incorporated hereinby reference.

GOVERNMENT SUPPORT

The invention was supported in part by Grant No.: 08748 from theNational Cancer Institute; Grant Nos.: AI-16943 and GM056904 from theNational Institutes of Health; and by Postdoctoral Fellowships forChristoph Gaul (Deutscher Akademischer Austauschdienst, DAAD) and JonTryggvi Njardarson (General Motors Cancer Research Program). The U.S.government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Migrastatin (1) is a novel 14-membered ring macrolide natural product,that was first isolated from a cultured broth of Steptomyces sp.MK929-43F1 by Imoto et al. (see, Nakae et al., J. Antibiot., 2000, 53,1130-1136; and Nakae et al., J. Antibiot., 2000, 53, 1228-1230). It wasrecently reported that cultures of Steptomyces platensis also produceMigrastatin (see, Woo et al., J. Antibiot., 2002, 55, 141-146).

Migrastatin has been shown to inhibit both migration andanchorage-independent growth of human tumor cells (see, Nakae et al., J.Antibiot., 2001, 54, 1104-1107), and has sparked interest in the area ofcancer research. Specifically, migration of tumor cells is part of thecomplex process of metastasis, which is the leading cause of death incancer patients. Therefore, Migrastatin and derivatives thereof holdgreat potential as therapeutic agents for the treatment of cancer.

After initial isolation and reporting of this compound, several groupsexplored the possibility of preparing derivatives and/or furtherexploring their biological activity. Each of these groups, however, wasonly able to obtain Migrastatin and derivatives thereof by fermentationtechniques and/or by modifications to the natural product, and thus waslimited in the number and types of derivatives that could be preparedand/or evaluated for biological activity.

Clearly, there remains a need for compounds related to Migrastatin.Therefore, there is a need to develop synthetic methodologies to accessa variety of novel analogues of Migrastatin, particularly those that areinaccessible by making modifications to the natural product. It wouldalso be of particular interest to develop novel compounds that exhibit afavorable therapeutic profile in vivo (e.g., are safe and effective).

SUMMARY OF THE INVENTION

In one aspect, the present invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of a compoundof general formula (I),

as described generally and in subclasses herein, whereby the compositionis formulated for administration to a subject at a dosage between about0.1 mg/kg to about 50 mg/kg of body weight.

In another aspect, the present invention provides a method for treatingbreast tumor metastasis in a subject comprising administering to asubject in need thereof a therapeutically effective amount of theinventive composition described directly above and a pharmaceuticallyacceptable carrier, adjuvant or vehicle.

DEFINITIONS

The term “aliphatic”, as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched) or branched aliphatichydrocarbons, which are optionally substituted with one or morefunctional groups. As will be appreciated by one of ordinary skill inthe art, “aliphatic” is intended herein to include, but is not limitedto, alkyl, alkenyl, alkynyl moieties. Thus, as used herein, the term“alkyl” includes straight and branched alkyl groups. An analogousconvention applies to other generic terms such as “alkenyl”, “alkynyl”and the like. Furthermore, as used herein, the terms “alkyl”, “alkenyl”,“alkynyl” and the like encompass both substituted and unsubstitutedgroups. In certain embodiments, as used herein, “lower alkyl” is used toindicate those alkyl groups (cyclic, acyclic, substituted,unsubstituted, branched or unbranched) having about 1-6 carbon atoms.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employedin the invention contain about 1-20 aliphatic carbon atoms. In certainother embodiments, the alkyl, alkenyl, and alkynyl groups employed inthe invention contain about 1-10 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-4 carbon atoms. Illustrative aliphatic groupsthus include, but are not limited to, for example, methyl, ethyl,n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl,moieties and the like, which again, may bear one or more substituents.Alkenyl groups include, but are not limited to, for example, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representativealkynyl groups include, but are not limited to, ethynyl, 2-propynyl(propargyl), 1-propynyl and the like.

The term “alicyclic”, as used herein, refers to compounds which combinethe properties of aliphatic and cyclic compounds and include but are notlimited to cyclic, or polycyclic aliphatic hydrocarbons and bridgedcycloalkyl compounds, which are optionally substituted with one or morefunctional groups. As will be appreciated by one of ordinary skill inthe art, “alicyclic” is intended herein to include, but is not limitedto, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, which areoptionally substituted with one or more functional groups. Illustrativealicyclic groups thus include, but are not limited to, for example,cyclopropyl, —CH₂-cyclopropyl, cyclobutyl, —CH₂-cyclobutyl, cyclopentyl,—CH₂-cyclopentyl-n, cyclohexyl, —CH₂-cyclohexyl, cyclohexenylethyl,cyclohexanylethyl, norborbyl moieties and the like, which again, maybear one or more substituents.

The terms “alkoxy” (or “alkyloxy”), and “thioalkyl” as used hereinrefers to an alkyl group, as previously defined, attached to the parentmolecular moiety through an oxygen atom (“alkoxy”) or through a sulfuratom (“thioalkyl”). In certain embodiments, the alkyl group containsabout 1-20 aliphatic carbon atoms. In certain other embodiments, thealkyl group contains about 1-10 aliphatic carbon atoms. In yet otherembodiments, the alkyl group contains about 1-8 aliphatic carbon atoms.In still other embodiments, the alkyl group contains about 1-6 aliphaticcarbon atoms. In yet other embodiments, the alkyl group contains about1-4 aliphatic carbon atoms. Examples of alkoxy groups, include but arenot limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,tert-butoxy, neopentoxy and n-hexoxy. Examples of thioalkyl groupsinclude, but are not limited to, methylthio, ethylthio, propylthio,isopropylthio, n-butylthio, and the like.

The term “alkylamino” refers to a group having the structure —NHR′wherein R′ is alkyl, as defined herein. The term “aminoalkyl” refers toa group having the structure NH₂R′—, wherein R′ is alkyl, as definedherein. In certain embodiments, the alkyl group contains about 1-20aliphatic carbon atoms. In certain other embodiments, the alkyl groupcontains about 1-10 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention containabout 1-8 aliphatic carbon atoms. In still other embodiments, the alkylgroup contains about 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl group contains about 1-4 aliphatic carbon atoms.Examples of alkylamino include, but are not limited to, methylamino,ethylamino, iso-propylamino and the like.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl, wherein any of the aliphatic, heteroaliphatic,alkylaryl, or alkylheteroaryl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples that are describedherein.

In general, the terms “aryl” and “heteroaryl”, as used herein, refer tostable mono- or polycyclic, heterocyclic, polycyclic, andpolyheterocyclic unsaturated moieties having preferably 3-14 carbonatoms, each of which may be substituted or unsubstituted. It will alsobe appreciated that aryl and heteroaryl moieties, as defined herein maybe attached via an aliphatic, alicyclic, heteroaliphatic,heteroalicyclic, alkyl or heteroalkyl moiety and thus also include-(aliphatic)aryl, -(heteroaliphatic)aryl, -(aliphatic)heteroaryl,-(heteroaliphatic)heteroaryl, and specifically -(alkyl)aryl,-(heteroalkyl)aryl, -(heteroalkyl)aryl, and -(heteroalkyl)heteroarylmoieties. Thus, as used herein, the phrases “aryl or heteroaryl” and“aryl, heteroaryl, -(aliphatic)aryl, -(heteroaliphatic)aryl,-(aliphatic)heteroaryl, -(heteroaliphatic)heteroaryl, -(alkyl)aryl,-(heteroalkyl)aryl, -(heteroalkyl)aryl, and -(heteroalkyl)heteroaryl”are often interchangeable. Substituents include, but are not limited to,any of the previously mentioned substitutents, i.e., the substituentsrecited for aliphatic moieties, or for other moieties as disclosedherein, resulting in the formation of a stable compound. In certainembodiments of the present invention, “aryl” refers to a mono- orbicyclic carbocyclic ring system having one or two aromatic ringsincluding, but not limited to, phenyl, naphthyl, tetrahydronaphthyl,indanyl, indenyl and the like. In certain embodiments of the presentinvention, the term “heteroaryl”, as used herein, refers to a cyclicaromatic radical having from about five to about ten ring atoms of whichone ring atom is selected from S, O and N; zero, one or two ring atomsare additional heteroatoms independently selected from S, O and N; andthe remaining ring atoms are carbon, the radical being joined to therest of the molecule via any of the ring atoms, such as, for example,pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl,furanyl, quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one, two or three of the hydrogenatoms thereon independently with any one or more of the followingmoieties including, but not limited to: aliphatic; heteroaliphatic;aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy;heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio;heteroarylthio; F; Cl; Br; I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂;—CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x));—CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂;—S(O)₂R_(x); —NR_(x)(CO)R_(x) wherein each occurrence of R_(x)independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl,wherein any of the aliphatic, heteroaliphatic, alkylaryl, oralkylheteroaryl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substituents are illustrated by the specificembodiments shown in the Examples that are described herein.

The term “cycloalkyl”, as used herein, refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof aliphatic, heteroaliphatic or heterocyclic moieties, may optionallybe substituted with substituents including, but not limited toaliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl, wherein any of the aliphatic, heteroaliphatic,alkylaryl, or alkylheteroaryl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples that are describedherein.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietiesin which one or more carbon atoms in the main chain have beensubstituted with a heteroatom. Thus, a heteroaliphatic group refers toan aliphatic chain which contains one or more oxygen, sulfur, nitrogen,phosphorus or silicon atoms, e.g., in place of carbon atoms.Heteroaliphatic moieties may be branched or linear unbranched. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;alicyclic; heteroaliphatic; heteroalicyclic; aryl; heteroaryl;alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F;Cl; Br; I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, alicyclic, heteroaliphatic, heteroalicyclic,aryl, heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of thealiphatic, alicyclic, heteroaliphatic, heteroalicyclic, alkylaryl, oralkylheteroaryl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substituents are illustrated by the specificembodiments shown in the Examples that are described herein.

The term “heteroalicyclic”, as used herein, refers to compounds whichcombine the properties of heteroaliphatic and cyclic compounds andinclude but are not limited to saturated and unsaturated mono- orpolycyclic heterocycles such as morpholino, pyrrolidinyl, furanyl,thiofuranyl, pyrrolyl etc., which are optionally substituted with one ormore functional groups, as defined herein.

Additionally, it will be appreciated that any of the alicyclic orheteroalicyclic moieties described above and herein may comprise an arylor heteroaryl moiety fused thereto. Additional examples of generallyapplicable substituents are illustrated by the specific embodimentsshown in the Examples that are described herein.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “acyloxy”, as used herein, does not substantially differ fromthe common meaning of this term in the art, and refers to a moiety ofstructure —OC(O)R_(x), wherein R_(x) is a substituted or unsubstituted,cyclic or acyclic, linear or branched, saturated or unsaturatedaliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl orheteroaryl moiety.

The term “acyl”, as used herein, does not substantially differ from thecommon meaning of this term in the art, and refers to a moiety ofstructure —C(O)R_(x), wherein R_(x) is a substituted or unsubstituted,cyclic or acyclic, linear or branched, saturated or unsaturatedaliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl orheteroaryl moiety.

The term “heterocycloalkyl” or “heterocycle”, as used herein, refers toa non-aromatic 5-, 6- or 7-membered ring or a polycyclic group,including, but not limited to a bi- or tri-cyclic group comprising fusedsix-membered rings having between one and three heteroatomsindependently selected from oxygen, sulfur and nitrogen, wherein (i)each 5-membered ring has 0 to 1 double bonds and each 6-membered ringhas 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may beoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above heterocyclic rings may be fusedto an aryl or heteroaryl ring. Representative heterocycles include, butare not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl,isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, andtetrahydrofuryl. In certain embodiments, a “substituted heterocycloalkylor heterocycle” group is utilized and as used herein, refers to aheterocycloalkyl or heterocycle group, as defined above, substituted bythe independent replacement of one, two or three of the hydrogen atomsthereon with but are not limited to aliphatic; heteroaliphatic; aryl;heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy;heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F;Cl; Br; I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH;—CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(X))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, oralkylheteroaryl, wherein any of the aliphatic, heteroaliphatic,alkylaryl, or alkylheteroaryl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substitutentsdescribed above and herein may be substituted or unsubstituted.Additional examples or generally applicable substituents are illustratedby the specific embodiments shown in the Examples, which are describedherein.

As used herein, the terms “aliphatic”, “heteroaliphatic”, “alkyl”,“alkenyl”, “alkynyl”, “heteroalkyl”, “heteroalkenyl”, “heteroalkynyl”,and the like encompass substituted and unsubstituted, saturated andunsaturated, and linear and branched groups. Similarly, the terms“alicyclic”, “heteroalicyclic”, “heterocycloalkyl”, “heterocycle” andthe like encompass substituted and unsubstituted, and saturated andunsaturated groups. Additionally, the terms “cycloalkyl”,“cycloalkenyl”, “cycloalkynyl”, “heterocycloalkyl”,“heterocycloalkenyl”, “heterocycloalkynyl”, “aryl”, “heteroaryl” and thelike encompass both substituted and unsubstituted groups.

The phrase, “pharmaceutically acceptable derivative”, as used herein,denotes any pharmaceutically acceptable salt, ester, or salt of suchester, of such compound, or any other adduct or derivative which, uponadministration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue thereof. Pharmaceutically acceptable derivatives thus includeamong others pro-drugs. A pro-drug is a derivative of a compound,usually with significantly reduced pharmacological activity, whichcontains an additional moiety, which is susceptible to removal in vivoyielding the parent molecule as the pharmacologically active species. Anexample of a pro-drug is an ester, which is cleaved in vivo to yield acompound of interest. Pro-drugs of a variety of compounds, and materialsand methods for derivatizing the parent compounds to create thepro-drugs, are known and may be adapted to the present invention.Certain exemplary pharmaceutical compositions and pharmaceuticallyacceptable derivatives will be discussed in more detail herein below.

By the term “protecting group”, has used herein, it is meant that aparticular functional moiety, e.g., O, S, or N, is temporarily blockedso that a reaction can be carried out selectively at another reactivesite in a multifunctional compound. In preferred embodiments, aprotecting group reacts selectively in good yield to give a protectedsubstrate that is stable to the projected reactions; the protectinggroup must be selectively removed in good yield by readily available,preferably nontoxic reagents that do not attack the other functionalgroups; the protecting group forms an easily separable derivative (morepreferably without the generation of new stereogenic centers); and theprotecting group has a minimum of additional functionality to avoidfurther sites of reaction. As detailed herein, oxygen, sulfur, nitrogenand carbon protecting groups may be utilized. For example, in certainembodiments, as detailed herein, certain exemplary oxygen protectinggroups are utilized. These oxygen protecting groups include, but are notlimited to methyl ethers, substituted methyl ethers (e.g., MOM(methoxymethyl ether), MTM (methylthiomethyl ether), BOM(benzyloxymethyl ether), PMBM or MPM (p-methoxybenzyloxymethyl ether),to name a few), substituted ethyl ethers, substituted benzyl ethers,silyl ethers (e.g., TMS (trimethylsilyl ether), TES(triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS(t-butyldimethylsilyl ether), tribenzyl silyl ether, TBDPS(t-butyldiphenyl silyl ether), to name a few), esters (e.g., formate,acetate, benzoate (Bz), trifluoroacetate, dichloroacetate, to name afew), carbonates, cyclic acetals and ketals. In certain other exemplaryembodiments, nitrogen protecting groups are utilized. These nitrogenprotecting groups include, but are not limited to, carbamates (includingmethyl, ethyl and substituted ethyl carbamates (e.g., Troc), to name afew) amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, iminederivatives, and enamine derivatives, to name a few. Certain otherexemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the present invention. Additionally, a variety of protectinggroups are described in “Protective Groups in Organic Synthesis” ThirdEd. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York:1999, the entire contents of which are hereby incorporated by reference.

As used herein, the term “reaction vessel” denotes any container thatcan contain a reacting solution. For example, test tubes, petri dishes,and wells can all constitute reaction vessels. Preferably, a reactionvessel is a well in a multiwell plate or other multivessel format.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A depicts a ¹H NMR spectrum of synthetic Migrastatin.

FIG. 1B depicts a ¹H NMR spectrum of naturally occurring Migrastatin.

FIG. 2 depicts effects of inventive compounds on 4T1 tumor cellmigration: (A) macrolactone 48; and (B) migrastatin (1).

FIG. 3 depicts effects of inventive compounds on 4T1 cell proliferation.

FIG. 4 depicts effects of treatment with exemplary migrastatin analogson 4T1 tumor lung metastasis in syngeneic mice.

FIG. 5 depicts effects of migrastatin analogs on 4T1 cell tumor growth.

FIG. 6 depicts effects of migrastatin analogs on wound healing. (A) noserum; (B) with serum; (C) Macrolactone 48 and serum (200 nM); and (D)Migrastatin (1) and serum (200 nM).

FIG. 7 depicts a crystallographic structure of dimer compound 24.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

In recognition of the need to access novel Migrastatin analogs, and thisclass of macrocycles in general, the present invention provides novelmacrocyclic compounds, as described in more detail herein, which exhibitthe ability to inhibit cell migration. Therefore, the compounds may beuseful as angiogenesis inhibitors. The invention also providesinformation regarding structural elements that participate in orcontribute to this activity, and therefore provides insight into thebiological activity of this class of compounds. Thus, the compounds ofthe invention, and pharmaceutical compositions thereof, are useful asantiangiogenesis agents for the treatment of cancer and/or abnormal cellproliferation. In certain embodiments, the compounds of the presentinvention can be used for the treatment of diseases and disordersincluding, but not limited to solid tumor cancers, metastasis, ocularangiogenic diseases, diabetic retinopathy, retinopathy of prematurity,corneal graft rejection, neovascular glaucoma, retrolental fibroplasias,rubeosis, solid tumors, blood born tumors, leukemias, tumor metastases,benign tumors, acoustic neuromas, neurofibromas, trachomas, pyogenicgranulomas, rheumatoid arthritis, psoriasis, Osler-Webber Syndrome,myocardial angiogenesis, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma, or wound granulation, to name a few.

1) General Description of Compounds of the Invention

In certain embodiments, the compounds of the invention include compoundsof the general formula (I) as further defined below:

pharmaceutically acceptable derivatives thereof;

wherein R₁ and R₂ are each independently hydrogen, halogen, —CN,—S(O)₁₋₂R^(1A), —NO₂, COR^(1A), —CO₂R^(1A), —NR^(1A)C(═O)R^(1B),—NR^(1A)C(═O)OR^(1B), —CONR^(1A)R^(1B), an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(1A);wherein W is independently —O—, —S— or —NR^(1C)—, wherein eachoccurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R₁ and R₂, taken together with the carbon atoms towhich they are attached, form an alicyclic, heteroalicyclic, aryl orheteroaryl moiety;

R₃ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group;

R₄ is halogen, —OR^(4A), —OC(═O)R^(4A) or —NR^(4A)R^(4B); wherein R^(4A)and R^(4B) are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; a prodrug moiety,a nitrogen protecting group or an oxygen protecting group; or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a heterocyclic or heteroaryl moiety; or R₄, takentogether with the carbon atom to which it is attached forms a moietyhaving the structure:

R₅ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), —NO₂, COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(6C)—, wherein each occurrence of R^(6A), R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R₆ and R_(c), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,—CN, —S(O)₁₂R^(a1), —NO₂, —COR^(a1), —CO₂R^(a1), —NR^(a1)C(═O)R^(a2),—NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2), an alphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(a1);wherein W is independently —O—, —S— or —NR^(a3)—, wherein eachoccurrence of R^(a1), R^(a2) and R^(a3) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —COR^(c1),—CO₂R^(c1), —NR^(c1)C(═O)R^(c2); —NR^(c1)C(═O)OR^(c2), —CONR^(c1)R^(c2);an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WW¹; wherein W is independently —O—, —S— or—NR^(c3)—, wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R_(c) and R₆, takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

n is an integer from 1 to 5;

X₁ is O, S, NR^(X1) or CR^(X1)R^(X2); wherein R^(X1) and R^(X2) areindependently hydrogen, halogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or a nitrogenprotecting group;

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

Y₁ and Y₂ are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; or —WR^(Y1);wherein W is independently —O—, —S— or —NR^(Y2)—, wherein eachoccurrence of R^(Y1) and R^(Y2) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or Y₁ and Y₂ together with the carbon atom to whichthey are attached form a moiety having the structure:

In certain embodiments of compounds described directly above andcompounds as described in certain classes and subclasses herein,inventive compounds do not have one of the following structures:

In certain other embodiments, compounds of formula (I) have thefollowing stereochemistry:

In certain other embodiments, compounds of formula (I) have thefollowing stereochemistry:

In certain other embodiments, compounds of formula (I) have thefollowing stereochemistry:

In certain other embodiments, compounds of formula (I) are defined asfollows:

R₁ and R₂ are each independently hydrogen or substituted orunsubstituted lower alkyl; or R₁ and R₂, taken together with the carbonatoms to which they are attached, form an epoxide, an aziridine or asubstituted or unsubstituted cyclopropyl moiety;

R₃ is hydrogen, or substituted or unsubstituted lower alkyl or aryl; aprodrug moiety or an oxygen protecting group;

R₄ is halogen, —OR^(4A), —OC(═O)R^(4A) or —NR^(4A)R^(4B); wherein R^(4A)and R^(4B) are independently hydrogen, or substituted or unsubstitutedlower alkyl; a prodrug moiety, a nitrogen protecting group or an oxygenprotecting group; or R^(4A) and R^(4B), taken together with the nitrogenatom to which they are attached, form a heterocyclic or heteroarylmoiety; or R₄, taken together with the carbon atom to which it isattached forms a moiety having the structure:

R₅ and R₆ are each independently hydrogen or substituted orunsubstituted lower alkyl; or R₆ and R_(c), taken together with thecarbon atoms to which they are attached, form an epoxide, an aziridineor a substituted or unsubstituted cyclopropyl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety, or —WR^(a1); wherein W is independently —O—, —S— or —NR^(a3)—,wherein each occurrence of R^(a1), and R^(a1) is independently hydrogen,or an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether, form an epoxide, an aziridine or a substituted orunsubstituted cyclopropyl moiety;

R_(c) is hydrogen, halogen, alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl moiety, or —WR^(c1); wherein W isindependently —O—, —S— or —NR^(c3)—, wherein each occurrence of R^(c1)and R^(c3) is independently hydrogen, or an alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl or heteroaryl moiety; or R_(c), andR₆, taken together with the carbon atoms to which they are attached,form an epoxide, an aziridine or a substituted or unsubstitutedcyclopropyl moiety;

n is an integer from 1 to 5;

X₁ is O, S, NR^(X1) or CR^(X1)R^(X2); wherein R^(X1) and R^(X2) areindependently hydrogen, halogen, substituted or unsubstituted alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, or anitrogen protecting group;

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

Y₁ and Y₂ are independently hydrogen, an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl moiety; or —WR^(Y1); wherein W isindependently —O—, —S— or —NR^(Y2)—, wherein each occurrence of R¹ andR² is independently hydrogen, or an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl moiety; or Y₁ and Y₂ together withthe carbon atom to which they are attached form a moiety having thestructure:

In certain embodiments, the present invention defines certain classes ofcompounds which are of special interest. For example, one class ofcompounds of special interest includes those compounds having thestructure of formula (I) in which R_(a), R_(b) and R_(c) are eachhydrogen, and the compound has one of the following structures:

wherein R₁-R₆, Y₂, X₁, n and Q are as defined in classes and subclassesherein; W is O or NH; and R^(Y1) is hydrogen, or an aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aryl or heteroaryl moiety.

In certain exemplary embodiments, compounds of the invention showndirectly above have the following stereochemistry:

Another class of compounds of special interest includes those compoundshaving the structure of formula (I) in which R_(a), R_(b) and R_(c) areeach hydrogen, Q is a carbonyl-containing moiety and the compound hasone of the following structures:

wherein R₁-R₆, Y₂, X₁, n and Q are as defined in classes and subclassesherein; W is O or NH; and R^(Y1) is hydrogen, or an aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aryl or heteroaryl moiety.

In certain exemplary embodiments, compounds of the invention showndirectly above have the following stereochemistry:

Another class of compounds of special interest includes compounds havingthe structure of formula (I) in which R_(a), R_(b) and R_(c) are eachhydrogen, n is 3 and the compound has one of the following structures:

wherein R₁-R₆, Y₂, Q and X₁ are as defined in classes and subclassesherein; W is O or NH; and R^(Y1) is hydrogen, or an aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aryl or heteroaryl moiety.

In certain exemplary embodiments, compounds of the invention showndirectly above have the following stereochemistry:

Another class of compounds of special interest includes compounds havingthe structure of formula (I) in which R_(a), R_(b) and R_(c) are eachhydrogen, n is 3, Q is a carbonyl-containing moiety, and the compoundhas one of the following structures:

wherein R₁-R₆, X₁ and Y₂ are as defined in classes and subclassesherein; W is O or NH; R^(Y1) is hydrogen, or an aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aryl or heteroaryl moiety;R₇ is a substituted or unsubstituted lower alkyl or heteroalkyl moiety;R₈ is a substituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl moiety; and Alk is a substituted orunsubstituted C₀₋₆alkylidene or C₀₋₆alkenylidene chain wherein up to twonon-adjacent methylene units are independently optionally replaced byCO, CO₂, COCO, CONR^(Z1), OCONR^(Z1), NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO,NR^(Z1)CO, NR^(Z1)CO₂, NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂,SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein each occurrenceof and R^(Z2) is independently hydrogen, alkyl, heteroalkyl, aryl,heteroaryl or acyl.

In certain exemplary embodiments, compounds of the invention showndirectly above have the following stereochemistry:

Another class of compounds of special interest includes those compoundshaving the structure of formula (I) in which R_(a), R_(b) and R_(c) areeach hydrogen, Q is hydrogen and the compound has the followingstructure:

wherein R₁-R₆, Y₁, Y₂, X₁, and n are as defined in classes andsubclasses herein.

In certain exemplary embodiments, compounds of the invention showndirectly above have the following stereochemistry:

Another class of compounds of special interest includes compounds havingthe structure of formula (I) in which R_(a), R_(b) and R_(c) are eachhydrogen, Q is hydrogen, n is 3 and the compound has the followingstructure:

wherein R₁-R₆, Y₁, Y₂, and X₁ are as defined in classes and subclassesherein.

In certain exemplary embodiments, compounds of the invention showndirectly above have the following stereochemistry:

The following structures illustrate several exemplary types of compoundsof these classes. Additional compounds are described in theExemplification herein. Other compounds of the invention will be readilyapparent to the reader:

A number of important subclasses of each of the foregoing classesdeserve separate mention; these subclasses include subclasses of theforegoing classes in which:

i) R₁ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(1A), —NO₂, —COR^(1A),—CO₂R^(1A), —NR^(1A)C(═O)OR^(1B), CONR^(1A)R^(1B), an aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aryl or heteroaryl moiety,or —WR^(1A); wherein W is independently —O—, —S— or —NR^(1C)—, whereineach occurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen,or an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety;

ii) R₁ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(1A), —NO₂, —COR^(1A),—CO₂R^(1A), —NR^(1A)C(═O)R^(1B), —NR^(1A)C(═O)OR^(1B), —CONR^(1A)R^(1B),an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety, or —WR^(1A); wherein W is independently —O—, —S— or —NR^(1C)—,wherein each occurrence of R^(1A), R^(1B) and R^(1C) is independentlyhydrogen, or an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, arylor heteroaryl moiety;

iii) R₁ is hydrogen or lower alkyl;

iv) R₁ is hydrogen;

v) R₂ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(1A), —NO₂, —COR^(1A),—CO₂R_(1A), —NR^(1A)C(═O)R^(1B), —NR^(1A)C(═O)OR^(1B), CONR^(1A)R^(1B),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(1A); wherein W is independently —O—, —S— or—NR^(1C)—, wherein each occurrence of R^(1A), R^(1B) and R^(1C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

vi) R₂ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(1A), —NO₂, —COR^(1A),—CO₂R^(1A), —NR^(1A)C(═O)R^(1B), —NR^(1A)C(═O)OR^(1B), —CONR^(1A)R^(1B),an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety, or —WR^(1A); wherein W is independently —O—, —S— or —NR^(1C)—,wherein each occurrence of R^(1A), R^(1B) and R^(1C) is independentlyhydrogen, or an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, arylor heteroaryl moiety;

vii) R₂ is hydrogen or lower alkyl;

viii) R₂ is hydrogen;

ix) R₁ and R₂ are each hydrogen;

x) R₁ and R₂, taken together with the carbon atoms to which they areattached, form an alicyclic, heteroalicyclic, aryl or heteroaryl moiety;

xi) R₁ and R₂, taken together with the carbon atoms to which they areattached, form a cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety;

xii) R₁ and R₂, taken together with the carbon atoms to which they areattached, form an epoxide;

xiii) R₁ and R₂, taken together with the carbon atoms to which they areattached, form an aziridine;

xiv) R₁ and R₂, taken together with the carbon atoms to which they areattached, form a substituted or unsubstituted cyclopropyl;

XV) R₃ is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, aryl, heteroaryl, silyl, —C(═O)R^(x), —C(═S)R^(x),—C(═NR^(x))R^(y), —SO₂R^(x), wherein R^(x) and R^(y) are eachindependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aryl, heteroaryl, —C(═O)R^(A) or-ZR^(A), wherein Z is —O—, —S—, —NR^(B), wherein each occurrence ofR^(A) and R^(B) is independently hydrogen, or an alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroarylmoiety;

xvi) R₃ is hydrogen, an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group;

xvii) R₃ is hydrogen, lower alkyl, aryl, a prodrug moiety or an oxygenprotecting group;

xviii) R₃ is hydrogen, lower alkyl, aryl or an oxygen protecting group;

xix) R₃ is methyl;

xxi) the carbon atom bearing R₄ is of R-configuration; xxii) the carbonatom bearing R₄ is of S-configuration

xxiii) R₄ is halogen, —OR^(4A), —OC(═O)R^(4A) or —NR^(4A)R^(4B); whereinR^(4A) and R^(4B) are independently hydrogen, or substituted orunsubstituted lower alkyl; a prodrug moiety, a nitrogen protecting groupor an oxygen protecting group; or R^(4A) and R^(4B), taken together withthe nitrogen atom to which they are attached, form a heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

xxiv) R₄ is a halogen selected from fluorine, chlorine, bromine, andiodine;

xxv) R₄ is fluorine;

xxvi) the carbon atom bearing R₄ is of R-configuration, and R₄ is ahalogen selected from fluorine, chlorine, bromine, and iodine;

xxvii) the carbon atom bearing R₄ is of R-configuration, and R₄ isfluorine;

xxviii) R₄ is OR^(4A), wherein R^(4A) is hydrogen, a substituted orunsubstituted lower alkyl; acyl; a prodrug moiety or an oxygenprotecting group;

xxix) R₄ is OH;

xxx) R₄ is —OC(═O)R^(4A) wherein R^(4A) is hydrogen, lower alkyl, arylor heteroaryl;

xxxi) R₄ is OAc;

xxxii) R₄ is NR^(4A)R^(4B); wherein R^(4A) and R^(4B) are independentlyhydrogen, a substituted or unsubstituted lower alkyl; a prodrug moietyor a nitrogen protecting group; or R^(4A) and R^(4B), taken togetherwith the nitrogen atom to which they are attached, form a heterocyclicor heteroaryl moiety;

xxxiii) R₄ is NR^(4A)R^(4B); wherein R^(4A) and R^(4B) are independentlyhydrogen, alkyl, alkenyl, —C(═O)R^(x), —C(═O)OR^(x), —SR^(x), SO₂R^(x),or R^(4A) and R^(4B), taken together with the nitrogen atom to whichthey are attached form a moiety having the structure ═CR^(x)R^(y),wherein R^(4A) and R^(4B) are not simultaneously hydrogen and R^(x) andR^(y) are each independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aryl, heteroaryl, —C(═O)R^(A) or-ZR^(A), wherein Z is —O—, —S—, —NR^(B), wherein each occurrence ofR^(A) and R^(B) is independently hydrogen, or an alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroarylmoiety;

xxxiv) R₄ is NH₂;

xxxv) R₄ together with the carbon atom to which it is attached forms amoiety having the structure:

xxxvi) R₄ together with the carbon atom to which it is attached forms amoiety having the structure:

xxxvii) R₅ is hydrogen or an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl moiety;

xxxviii) R₅ is hydrogen or substituted or unsubstituted lower alkyl;

xxxix) R₅ is methyl;

xl) R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), —NO₂, —COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), CONR^(6A)R^(6B),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(1C)—, wherein each occurrence of R^(6A), R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

xli) R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), —NO₂, —COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B),an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety, or —WR^(6A); wherein W is independently —O—, —S— or —NR^(1C)—,wherein each occurrence of R^(6A)R^(6B) and R^(6C) is independentlyhydrogen, or an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, arylor heteroaryl moiety;

xlii) R₆ is hydrogen or substituted or unsubstituted lower alkyl;

xliii) R₆ is methyl;

xxliv) R₅ and R₆ are each methyl;

xlv) R_(a) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(a1), —NO₂, —COR^(a1),—CO₂R^(a1), —NR^(a1)C(═O)R^(a2), —NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(a1); wherein W is independently —O—, —S— or—NR^(1C)—, wherein each occurrence of R^(a1), R^(a2) and R^(a3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

xlvi) R_(a) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(a1), —NO₂, —COR^(a1),—CO₂R^(a1), —NR^(a1)C(═O)R^(a2), —NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2),an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety, or —WR^(a1); wherein W is independently —O—, —S— or —NR^(1C)—,wherein each occurrence of R^(a1), R^(a2) and R^(a3) is independentlyhydrogen, or an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, arylor heteroaryl moiety;

xlvii) R_(a) is hydrogen or lower alkyl;

xlviii) R_(a) is hydrogen;

xlix) R_(b) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(a1), —NO₂, —COR^(a1),—CO₂R^(a1), —NR^(a1)C(═O)R^(a2), —NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(a1); wherein W is independently —O—, —S— or—NR^(1C)—, wherein each occurrence of R^(a1), R^(a2) and R^(a3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

1) R_(b) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(a1), —NO₂, —COR^(a1),—CO₂R^(a1), —NR^(a1)C(═O)R^(a2), —NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2),an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety, or —WR^(a1); wherein W is independently —O—, —S— or —NR^(1C)—,wherein each occurrence of R^(a1), R^(a2) and R^(a3) is independentlyhydrogen, or an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, arylor heteroaryl moiety;

li) R_(b) is hydrogen or lower alkyl;

lii) R_(b) is hydrogen;

liii) R_(a) and R_(b) are each hydrogen;

liv) R_(a) and R_(b), taken together with the carbon atoms to which theyare attached, form a cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety;

lv) R_(a) and R_(b), taken together with the carbon atoms to which theyare attached, form an epoxide;

lvi) R_(a) and R_(b), taken together with the carbon atoms to which theyare attached, form an aziridine;

lvii) R_(a) and R_(b), taken together with the carbon atoms to whichthey are attached, form a substituted or unsubstituted cyclopropyl;

lviii) R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —COR^(c1),—CO₂R^(c1), —NR^(c1)C(═O)R^(c2), —NR^(a1)C(═O)OR^(c2), —CONR^(c1)R^(c2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WW¹; wherein W is independently —O—, —S— or—NR^(1C), wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

lix) R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —COR^(c1),—CO₂R^(c1), —NR^(c1)C(═O)R^(c2), —NR^(c1)C(═O)OR^(c2), —CONR^(c1)R^(c2),an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety, or —WR^(c1); wherein W is independently —O—, —S— or —NR^(1C)—,wherein each occurrence of R^(c1), R^(c2) and R^(c3) is independentlyhydrogen, or an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, arylor heteroaryl moiety;

lx) R_(c) is hydrogen or lower alkyl;

lxi) R_(c) is hydrogen;

lxii) R_(c) and R₆, taken together with the carbon atoms to which theyare attached, form a cycloalkyl, heterocycloalkyl, aryl or heteroarylmoiety;

lxiii) R_(c) and R₆, taken together with the carbon atoms to which theyare attached with the carbon atoms to which they are attached, form anepoxide;

lxiv) R_(c) and R₆, taken together with the carbon atoms to which theyare attached, form an aziridine;

lxv) R_(c) and R₆, taken together with the carbon atoms to which theyare attached, form a substituted or unsubstituted cyclopropyl;

lxvi) X₁ is O, S, NR^(X1) or CR^(X1)R^(X2); wherein R^(X1) and R^(X2)are independently hydrogen, halogen, substituted or unsubstituted alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, or anitrogen protecting group;

lxvii) X₁ is O, NR^(X1) or CR^(X1)R^(X2); wherein R^(X1) and R^(X2) areindependently hydrogen, halogen, substituted or unsubstituted alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, or anitrogen protecting group;

lxviii) X₁ is O;

lxix) X₁ is NH;

lxx) X₁ is CH₂;

lxxi) n is an integer from 1 to 5;

lxxii) n is 3;

lxxiii) Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),a substituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl; or —WR^(Q1); wherein W isindependently —O—, —S— or —NR^(Q3)—, wherein each occurrence of R^(Q1),R^(Q2) and R^(Q3) is independently hydrogen, or an alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl or heteroaryl moiety

lxxiv) Q is a substituted or unsubstituted carbonyl-containing alkyl orheteroalkyl moiety;

lxxv) Q comprises a carbonyl linked to a carbocyclic, heterocyclic, arylor heteroaryl moiety through a C₀₋₆alkylidene or C₀₋₆alkenylidene chainwherein up to two non-adjacent methylene units are independentlyoptionally replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O,S, or NR^(Z1); wherein each occurrence of R^(Z1) and R^(Z2) isindependently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl;

lxxvi) Q has the structure:

wherein R₇ is a substituted or unsubstituted lower alkyl or heteroalkylmoiety; R₈ is a substituted or unsubstituted alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl or heteroaryl moiety; and Alk is asubstituted or unsubstituted C₀₋₆alkylidene or C₀₋₆alkenylidene chainwherein up to two non-adjacent methylene units are independentlyoptionally replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O,S, or NR^(Z1); wherein each occurrence of R^(Z1) and R^(Z2) isindependently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl;

lxxvii) Q has the structure:

wherein R₇ is a substituted or unsubstituted, linear or branched, cyclicor acyclic lower alkyl moiety; R₈ is a substituted or unsubstitutedcarbocyclic, heterocyclic, aryl or heteroaryl moiety; and Alk is asubstituted or unsubstituted C₀₋₆alkylidene or C₀₋₆alkenylidene chainwherein up to two non-adjacent methylene units are independentlyoptionally replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O,S, or NR^(Z1); wherein each occurrence of R^(Z1) and R^(Z2) isindependently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl;

lxxxviii) Q has the structure:

wherein R₇ is a substituted or unsubstituted, linear or branched, cyclicor acyclic lower alkyl moiety; R₈ is a substituted or unsubstitutedcarbocyclic, heterocyclic, aryl or heteroaryl moiety; and X, Y and Z areindependently a bond, —O—, —S—, —C(═O)—, —NR^(Z1)—, —CHOR^(Z1),—CHNR^(Z1)R^(Z2), C═S, C═N(R^(Y1)) or —CH(Hal); or a substituted orunsubstituted C₀₋₆alkylidene or C₀₋₆alkenylidene chain wherein up to twonon-adjacent methylene units are independently optionally replaced byCO, CO₂, COCO, CONR^(Z1), OCONR^(Z1), NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO,NR^(Z1)CO, NR^(Z1)CO₂, NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂,SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein Hal is ahalogen selected from F, Cl, Br and I; and each occurrence of R^(Z1) andR^(Z2) is independently hydrogen, alkyl, heteroalkyl, aryl, heteroarylor acyl; or R^(Z1) and R^(Z2), taken together with the nitrogen atom towhich they are attached, for a heterocyclic or heteroaryl moiety;

lxxix) Q has the structure:

wherein R₇ is a substituted or unsubstituted, linear or branched, cyclicor acyclic lower alkyl moiety; R₈ is a substituted or unsubstitutedcarbocyclic, heterocyclic, aryl or heteroaryl moiety; and Y is a bond,—O—, —S—, —C(═O)—, —NR^(Z1)—, —CHOR^(Z1), —CHNR^(Z1)R^(Z2), C═S,C═N(R^(Y1)) or —CH(Hal); or a substituted or unsubstitutedC₀₋₆alkylidene or C₀₋₆alkenylidene chain wherein up to two non-adjacentmethylene units are independently optionally replaced by CO, CO₂, COCO,CONR^(Z1), OCONR^(Z1), NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO₂,NR^(Z1)CO₂, NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1),NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein Hal is a halogen selectedfrom F, Cl, Br and I; and each occurrence of R^(Z1) and R^(Z2) isindependently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl; orR^(Z1) and R^(Z2), taken together with the nitrogen atom to which theyare attached, for a heterocyclic or heteroaryl moiety;

lxxx) Q has the structure:

wherein R₇ is a substituted or unsubstituted, linear or branched, cyclicor acyclic lower alkyl moiety; R₈ is a substituted or unsubstitutedcarbocyclic, heterocyclic, aryl or heteroaryl moiety; and R^(Y) ishydrogen, halogen, —OR^(Y1) or —NR^(Y1)NR^(Y2); wherein R^(Y1) andR^(Y2) are independently hydrogen, alkyl, heteroalkyl, aryl, heteroarylor acyl, or R^(Y1) and R^(Y2), taken together with the nitrogen atom towhich they are attached, for a heterocyclic or heteroaryl moiety;

lxxxi) Q is hydrogen;

lxxxii) compounds of subsets lxxvi)-lxxx) wherein R₇ is substituted orunsubstituted lower alkyl;

lxxxiii) compounds of subsets lxxvi)-lxxx) wherein R₇ is methyl;

lxxxiv) compounds of subset lxxx) wherein R^(Y) is hydrogen;

lxxxv) compounds of subset lxxx) wherein R^(Y) is a halogen selectedfrom fluorine, chlorine, bromine, and iodine;

lxxxvi) compounds of subset lxxx) wherein R^(Y) is fluorine;

lxxxvii) compounds of subset lxxx) wherein R^(Y) is OR^(Y1), whereinR^(Y1) is hydrogen, a substituted or unsubstituted lower alkyl; aprodrug moiety or an oxygen protecting group;

lxxxviii) compounds of subset lxxx) wherein R^(Y) is OH;

lxxxix) compounds of subset lxxx) wherein R^(Y) is NR^(Y1)R^(Y2);wherein R¹ and R² are independently hydrogen, a substituted orunsubstituted lower alkyl; a prodrug moiety or a nitrogen protectinggroup; or R¹ and R², taken together with the nitrogen atom to which theyare attached, form a heterocyclic or heteroaryl moiety;

xc) compounds of subset lxxx) wherein R^(Y) is NH₂;

xci) compounds of subsets lxxvi)-lxxx) wherein R₈ is one of:

wherein p is an integer from 0 to 5; q is 1 or 2, r is an integer from 1to 6; each occurrence of R^(8A) is independently hydrogen, alkyl,heteroalkyl, aryl, heteroaryl, -(alkyl)aryl or -(alkyl)heteroaryl,—OR^(8C), —SR^(8C), —N(R^(8C))₂, —SO₂N(R^(8C))₂, —(C═O)N(R^(8C))₂,halogen, —CN, —NO₂, —(C═O)OR^(8C), —N(R^(8C))(C═O)R^(8D), wherein eachoccurrence of R^(8C) and R^(8D) is independently hydrogen, lower alkyl,lower heteroalkyl, aryl, heteroaryl, -(alkyl)aryl or -(alkyl)heteroaryl;and each occurrence of R^(8B) is independently hydrogen or lower alkyl;

xcii) compounds of subsets lxxvi)-lxxx) wherein R₈ is substituted orunsubstituted cycloalkyl;

xciii) compounds of subsets lxxvi)-lxxx) wherein R₈ is substituted orunsubstituted cyclohexyl;

xciv) compounds of subsets lxxvi)-lxxx) wherein R₈ has the structure:

wherein R^(8B) is hydrogen or lower alkyl;

xcv) compounds of subsets lxxvi)-lxxx) wherein R₈ has the structure:

wherein R^(8B) is hydrogen or methyl;

xcvi) compounds of subsets lxxvi)-lxxx) wherein R₈ has the structure:

xcvii) X₁ is O, CH₂ or NH; Q is as described in subsets lxxvi)-lxxx)wherein R₈ has the structure:

xcviii) Y₁ is OR^(Y1) and Y₂ is lower alkyl; wherein R^(Y1) is hydrogenor lower alkyl;

xcix) Y₁ is OR^(Y1) and Y₂ is lower alkyl substituted with one or morehalogen atoms selected from F, Cl, Br and I; wherein R^(Y1) is hydrogenor lower alkyl;

c) Y₁ is OH and Y₂ is CF₃;

ci) X₁ is CH₂; Y₁ is OR^(Y1) and Y₂ is lower alkyl; wherein R^(Y1) ishydrogen or lower alkyl;

cii) X₁ is CH₂; Y₁ is OR^(Y1) and Y₂ is lower alkyl substituted with oneor more halogen atoms selected from F, Cl, Br and I; wherein R^(Y1) ishydrogen or lower alkyl;

ciii) X₁ is CH₂; Y₁ is OH and Y₂ is CF₃;

civ) Y₁ and Y₂ together with the carbon atom to which they are attachedform a moiety having the structure:

cv) Y₁ and Y₂ together with the carbon atom to which they are attachedform a moiety having the structure:

wherein R^(Y1) and R^(Y2) are independently hydrogen or lower alkyl;

cvi) Y₁ and Y₂ together with the carbon atom to which they are attachedform a moiety having the structure:

wherein R^(Y1) is hydrogen or lower alkyl;

cvii) Y₁ and Y₂ together with the carbon atom to which they are attachedform a moiety having the structure:

wherein R^(Y1) is hydrogen or lower alkyl;

cviii) Y₁ and Y₂ together with the carbon atom to which they areattached form a moiety having the structure:

wherein R^(Y1) is hydrogen or lower alkyl;

cix) X₁ is O; and Y₁ and Y₂ together with the carbon atom to which theyare attached form a moiety having the structure:

cx) X₁ is NH; and Y₁ and Y₂ together with the carbon atom to which theyare attached form a moiety having the structure:

cxi) X₁ is CH₂; and Y₁ and Y₂ together with the carbon atom to whichthey are attached form a moiety having the structure:

cxii) X₁ is CH₂; and Y₁ and Y₂ together with the carbon atom to whichthey are attached form a moiety having the structure:

wherein R^(Y1) is hydrogen or lower alkyl;

cxiii) X₁ is CH₂; and Y₁ and Y₂ together with the carbon atom to whichthey are attached form a moiety having the structure:

; wherein R^(Y1) is hydrogen or lower alkyl;

cxiv) compounds as described in classes and subclasses herein whereinthe stereocenter

has the following stereochemistry

and/or

cxv) compounds as described in classes and subclasses herein wherein thestereocenter

has the following stereochemistry

It will be appreciated that for each of the classes and subclassesdescribed above and herein, any one or more occurrences of groups suchas aliphatic, heteroaliphatic, alkyl, heteroalkyl may independently besubstituted or unsubstituted, linear or branched, saturated orunsaturated; and any one or more occurrences of alicyclic, heterocyclic,cycloalkyl, aryl, heteroaryl, cycloaliphatic, cycloheteroaliphatic maybe substituted or unsubstituted.

The reader will also appreciate that all possible combinations of thevariables described in i)-through cxv) above (e.g., R₁-R₆, R_(a-c), n,Q, X₁, Y₁ and Y₂, among others) are considered part of the invention.Thus, the invention encompasses any and all compounds of formula I, andsubclasses thereof, generated by taking any possible permutation ofvariables R₁-R₆, R_(a-c), n, Q, X₁, Y₁ and Y₂, and othervariables/substituents (e.g., X, Y, Z, R^(Y), etc.) as further definedfor R₁-R₆, R_(a-c), n, Q, X₁, Y₁ and Y₂, described in i)-through cxv)above.

As the reader will appreciate, compounds of particular interest include,among others, those which share the attributes of one or more of theforegoing subclasses. Some of those subclasses are illustrated by thefollowing sorts of compounds:

I) Compounds of the Formula (and Pharmaceutically Acceptable DerivativesThereof):

wherein R₃-R₆, n and Q are as defined in classes and subclasses herein;and Y₂ and R^(Y1) are independently hydrogen or lower alkyl. In certainembodiments, R₃ is hydrogen, lower alkyl or an oxygen protecting group.In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, n is 3. In certain embodiments, R₄ is halogen, hydroxyl,lower alkoxy, acyloxy or NR^(4A)R^(4B), wherein R^(4A) and R^(4B) areindependently hydrogen, lower alkyl, aryl, acyl or a nitrogen protectinggroup, or R^(4A) and R^(4B), taken together with the nitrogen atom towhich they are attached, form a substituted or unsusbstitutedheterocyclic or heteroaryl moiety; or R₄, taken together with the carbonatom to which it is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain exemplary embodiments, Q is hydrogen or a carbonyl-containingmoiety. In certain exemplary embodiments, Q is hydrogen. In certainexemplary embodiments, Y₂ is hydrogen or lower alkyl substituted withone or more halogen atoms selected from F, Cl, Br and I. In certainexemplary embodiments, Y₂ is hydrogen or methyl substituted with one ormore halogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or CF₃. In certain exemplary embodiments,R^(Y1) is hydrogen or lower alkyl. In certain exemplary embodiments,R^(Y1) is hydrogen or methyl. In certain exemplary embodiments, Y₂ isCF₃ and R^(Y1) is methyl.

II) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ and Q are as defined in classes and subclasses herein; andY₂ and R^(Y1) are independently hydrogen or lower alkyl. in certainembodiments, R₃ is hydrogen, lower alkyl or an oxygen protecting group.In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, R₄ is halogen, hydroxyl, lower alkoxy, acyloxy orNR^(4A)R^(4B), wherein R^(4A) and R^(4B) are independently hydrogen,lower alkyl, aryl, acyl or a nitrogen protecting group, or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a substituted or unsusbstituted heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain exemplary embodiments, Q is hydrogen or a carbonyl-containingmoiety. In certain exemplary embodiments, Q is hydrogen. In certainexemplary embodiments, Y₂ is hydrogen or lower alkyl substituted withone or more halogen atoms selected from F, Cl, Br and I. In certainexemplary embodiments, Y₂ is hydrogen or methyl substituted with one ormore halogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or CF₃. In certain exemplary embodiments,R^(Y1) is hydrogen or lower alkyl. In certain exemplary embodiments,R^(Y1) is hydrogen or methyl. In certain exemplary embodiments, Y₂ isCF₃ and R^(Y1) is methyl.

In certain other embodiments, for compounds of classes I)-II) above, Qis a substituted or unsubstituted carbonyl-containing alkyl orheteroalkyl moiety. In certain exemplary embodiments, Q comprises acarbonyl linked to a carbocyclic, heterocyclic, aryl or heteroarylmoiety through a C₀₋₆alkylidene or C₀₋₆alkenylidene moiety. In certainembodiments, Q has the structure:

wherein R₇ is a substituted or unsubstituted, linear or branched, cyclicor acyclic lower alkyl moiety; R₈ is a substituted or unsubstitutedcarbocyclic, heterocyclic, aryl or heteroaryl moiety; and Alk is asubstituted or unsubstituted C₀₋₆alkylidene or C₀₋₆alkenylidene chainwherein up to two non-adjacent methylene units are independentlyoptionally replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O,S, or NR^(Z1); wherein each occurrence of R^(Z1) and R^(Z2) isindependently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl;and R₈ is a substituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl or heteroaryl moiety. In certain embodiments, R₇is lower alkyl. In certain other embodiments, Alk is a C₃ alkylidenemoiety. In yet other embodiments, R₈ is one of:

wherein p is an integer from 0 to 5; q is 1 or 2, r is an integer from 1to 6; each occurrence of R^(8A) is independently hydrogen, alkyl,heteroalkyl, aryl, heteroaryl, -(alkyl)aryl or -(alkyl)heteroaryl,—OR^(8C), —SR^(8C), —N(R^(8C))₂, —SO₂N(R^(8C))₂, —(C═O)N(R^(8C))₂,halogen, —CN, —NO₂, —(C═O)OR^(8C), —N(R^(8C))(C═O)R^(8D), wherein eachoccurrence of R^(8C) and R^(8D) is independently hydrogen, lower alkyl,lower heteroalkyl, aryl, heteroaryl, -(alkyl)aryl or -(alkyl)heteroaryl;and each occurrence of R^(8B) is independently hydrogen or lower alkyl.In certain exemplary embodiments, R₈ has the structure:

wherein R^(8B) is hydrogen or lower alkyl. In certain exemplaryembodiments, R^(8B) is hydrogen. In certain exemplary embodiments, Q hasthe following stereochemistry:

III) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ and n are as defined in classes and subclasses herein; Y₂and R^(Y1) are independently hydrogen or lower alkyl; R₇ is asubstituted or unsubstituted, linear or branched, cyclic or acycliclower alkyl moiety; R^(8B) is hydrogen or lower alkyl; and X, Y and Zare independently a bond, —O—, —S—, —C(═O)—, —NR^(Z1)—, —CHOR^(Z1),—CHNR^(Z1)R^(Z2), C═S, C═N(R^(Y1)) or —CH(Hal); or a substituted orunsubstituted C₀₋₆alkylidene or C₀₋₆alkenylidene chain wherein up to twonon-adjacent methylene units are independently optionally replaced byCO, CO₂, COCO, CONR^(Z1), OCONR^(Z1), NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO,NR^(Z1)CO, NR^(Z1)CO₂, NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂,SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein Hal is ahalogen selected from F, Cl, Br and I; and each occurrence of and R^(Z2)is independently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl;or R^(Z1) and R^(Z2), taken together with the nitrogen atom to whichthey are attached, for a heterocyclic or heteroaryl moiety; andpharmaceutically acceptable derivatives thereof. In certain embodiments,R₃ is hydrogen, lower alkyl or an oxygen protecting group. In certainexemplary embodiments, R₃ is methyl. In certain other embodiments, R₅and R₆ are independently lower alkyl. In certain exemplary embodiments,R₅ and R₆ are each methyl. In certain embodiments, n is 3. In certainembodiments, R₄ is halogen, hydroxyl, lower alkoxy, acyloxy orNR^(4A)R^(4B), wherein R^(4A) and R^(4B) are independently hydrogen,lower alkyl, aryl, acyl or a nitrogen protecting group, or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a substituted or unsusbstituted heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain other embodiments, R₇ is methyl. In certain otherembodiments, X and Z are each CH₂ and Y is —CHOH, —CHNH₂ or —CHF. Incertain other embodiments, R^(8B) is hydrogen, methyl or ethyl. Incertain exemplary embodiments, Y₂ is hydrogen or lower alkyl substitutedwith one or more halogen atoms selected from F, Cl, Br and I. In certainexemplary embodiments, Y₂ is hydrogen or methyl substituted with one ormore halogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or CF₃. In certain exemplary embodiments,R^(Y1) is hydrogen or lower alkyl. In certain exemplary embodiments,R^(Y1) is hydrogen or methyl. In certain exemplary embodiments, Y₂ isCF₃ and R^(Y1) is methyl.

IV) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ are as defined in classes and subclasses herein; Y₂ andR^(Y1) are independently hydrogen or lower alkyl; R₇ is a substituted orunsubstituted, linear or branched, cyclic or acyclic lower alkyl moiety;R^(8B) is hydrogen or lower alkyl; and X, Y and Z are independently abond, —O—, —S—, —C(═O)—, —NR^(Z1)—, —CHOR^(Z1), —CHNR^(Z1)R^(Z2), C═S,C═N(R^(Y1)) or —CH(Hal); or a substituted or unsubstitutedC₀₋₆alkylidene or C₀₋₆alkenylidene chain wherein up to two non-adjacentmethylene units are independently optionally replaced by CO, CO₂, COCO,CONR^(Z1), OCONR^(Z1), NR^(Z1)NR^(Z2), NR^(Z1), NR^(Z2)CO, NR^(Z1)CO₂,NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O,S, or NR^(Z1); wherein Hal is a halogen selected from F, Cl, Br and I;and each occurrence of and R^(Z2) is independently hydrogen, alkyl,heteroalkyl, aryl, heteroaryl or acyl; or R^(Z1) and R^(Z2), takentogether with the nitrogen atom to which they are attached, for aheterocyclic or heteroaryl moiety; and pharmaceutically acceptablederivatives thereof. In certain embodiments, R₃ is hydrogen, lower alkylor an oxygen protecting group. In certain exemplary embodiments, R₃ ismethyl. In certain other embodiments, R₅ and R₆ are independently loweralkyl. In certain exemplary embodiments, R₅ and R₆ are each methyl. Incertain embodiments, R₄ is halogen, hydroxyl, lower alkoxy, acyloxy orNR^(4A)R^(4B), wherein R^(4A) and R^(4B) are independently hydrogen,lower alkyl, aryl, acyl or a nitrogen protecting group, or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a substituted or unsusbstituted heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain other embodiments, R₇ is methyl. In certain otherembodiments, X and Z are each CH₂ and Y is —CHOH, —CHNH₂ or —CHF. Incertain other embodiments, R^(8B) is hydrogen, methyl or ethyl. Incertain exemplary embodiments, Y₂ is hydrogen or lower alkyl substitutedwith one or more halogen atoms selected from F, Cl, Br and I. In certainexemplary embodiments, Y₂ is hydrogen or methyl substituted with one ormore halogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or CF₃. In certain exemplary embodiments,R^(Y1) is hydrogen or lower alkyl. In certain exemplary embodiments,R^(Y1) is hydrogen or methyl. In certain exemplary embodiments, Y₂ isCF₃ and R^(Y1) is methyl.

In certain embodiments, for compounds of classes III-IV above, —X—Y-Ztogether represents the moiety —CH₂—Y—CH₂—; wherein Y is —CHOR^(Y1),—CHNR^(Y1)R^(Y2), C═O, C═S, C═N(R^(Y1)) or —CH(Hal); wherein Hal is ahalogen selected from F, Cl, Br and I; and R^(Y1) and R^(Y2) areindependently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl, orR^(Y1) and R^(Y2), taken together with the nitrogen atom to which theyare attached, for a heterocyclic or heteroaryl moiety.

V) Compounds of the Formula (and Pharmaceutically Acceptable DerivativesThereof):

wherein R₃-R₆ and n are as defined in classes and subclasses herein; Y₂and R^(Y1) are independently hydrogen or lower alkyl; R₇ is asubstituted or unsubstituted, linear or branched, cyclic or acycliclower alkyl moiety; R^(8B) is hydrogen or lower alkyl; and Y is—CHOR^(Y1), CHNR^(Y1)R^(Y2), C═O, C═S, C═N(R^(Y1)) or —CH(Hal); whereinHal is a halogen selected from F, Cl, Br and I; and R^(Y1) and R^(Y2)are independently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl oracyl, or R^(Y1) and R^(Y2), taken together with the nitrogen atom towhich they are attached, for a heterocyclic or heteroaryl moiety. Incertain embodiments, R₃ is hydrogen, lower alkyl or an oxygen protectinggroup. In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, n is 3. In certain embodiments, R₄ is halogen, hydroxyl,lower alkoxy, acyloxy or NR^(4A)R^(4B), wherein R^(4A) and R^(4B) areindependently hydrogen, lower alkyl, aryl, acyl or a nitrogen protectinggroup, or R^(4A) and R^(4B), taken together with the nitrogen atom towhich they are attached, form a substituted or unsusbstitutedheterocyclic or heteroaryl moiety; or R₄, taken together with the carbonatom to which it is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain other embodiments, R₇ is methyl. In certain otherembodiments, Y is —CHOH, —CHNH₂ or —CHF. In certain other embodiments,R^(8B) is hydrogen, methyl or ethyl. In certain exemplary embodiments,Y₂ is hydrogen or lower alkyl substituted with one or more halogen atomsselected from F, Cl, Br and I. In certain exemplary embodiments, Y₂ ishydrogen or methyl substituted with one or more halogen atoms selectedfrom F, Cl, Br and I. In certain exemplary embodiments, Y₂ is hydrogenor CF₃. In certain exemplary embodiments, R^(Y1) is hydrogen or loweralkyl. In certain exemplary embodiments, R^(Y1) is hydrogen or methyl.In certain exemplary embodiments, Y₂ is CF₃ and R^(Y1) is methyl.

VI) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ are as defined in classes and subclasses herein; Y₂ andR^(Y1) are independently hydrogen or lower alkyl; R₇ is a substituted orunsubstituted, linear or branched, cyclic or acyclic lower alkyl moiety;R^(8B) is hydrogen or lower alkyl; and Y is —CHOR^(Y1),—CHNR^(Y1)R^(Y2), C═O, C═S, C═N(R^(Y1)) or —CH(Hal); wherein Hal is ahalogen selected from F, Cl, Br and I; and R^(Y1) and R^(Y2) areindependently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl, orR^(Y1) and R^(Y2), taken together with the nitrogen atom to which theyare attached, for a heterocyclic or heteroaryl moiety. In certainembodiments, R₃ is hydrogen, lower alkyl or an oxygen protecting group.In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, R₄ is halogen, hydroxyl, lower alkoxy, acyloxy orNR^(4A)R^(4B), wherein R^(4A) and R^(4B) are independently hydrogen,lower alkyl, aryl, acyl or a nitrogen protecting group, or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a substituted or unsusbstituted heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain other embodiments, R₇ is methyl. In certain otherembodiments, Y is —CHOH, —CHNH₂ or —CHF. In certain other embodiments,R^(8B) is hydrogen, methyl or ethyl. In certain exemplary embodiments,Y₂ is hydrogen or lower alkyl substituted with one or more halogen atomsselected from F, Cl, Br and I. In certain exemplary embodiments, Y₂ ishydrogen or methyl substituted with one or more halogen atoms selectedfrom F, Cl, Br and I. In certain exemplary embodiments, Y₂ is hydrogenor CF₃. In certain exemplary embodiments, R^(Y1) is hydrogen or loweralkyl. In certain exemplary embodiments, R^(Y1) is hydrogen or methyl.In certain exemplary embodiments, Y₂ is CF₃ and R^(Y1) is methyl.

VII) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein n, R₃ and R₄ are as defined in classes and subclasses herein; Y₂and R^(Y1) are independently hydrogen or lower alkyl; R^(8B) is hydrogenor lower alkyl; and R^(Y) is hydrogen, halogen, —OR^(Y1) or—NR^(Y1)NR^(Y2); wherein R^(Y1) and R^(Y2) are independently hydrogen,alkyl, heteroalkyl, aryl, heteroaryl or acyl, or R^(Y1) and R^(Y2),taken together with the nitrogen atom to which they are attached, for aheterocyclic or heteroaryl moiety. In certain embodiments, R₃ ishydrogen, lower alkyl or an oxygen protecting group. In certainexemplary embodiments, R₃ is methyl. In certain embodiments, n is 3. Incertain embodiments, R₄ is halogen, hydroxyl, lower alkoxy, acyloxy orNR^(4A)R^(4B), wherein R^(4A) and R^(4B) are independently hydrogen,lower alkyl, aryl, acyl or a nitrogen protecting group, or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a substituted or unsusbstituted heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain other embodiments, R^(Y) is OH, NH₂ or halogen (e.g., F). Incertain other embodiments, R^(8B) is hydrogen, methyl or ethyl. Incertain exemplary embodiments, Y₂ is hydrogen or lower alkyl substitutedwith one or more halogen atoms selected from F, Cl, Br and I. In certainexemplary embodiments, Y₂ is hydrogen or methyl substituted with one ormore halogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or CF₃. In certain exemplary embodiments,R^(Y1) is hydrogen or lower alkyl. In certain exemplary embodiments,R^(Y1) is hydrogen or methyl. In certain exemplary embodiments, Y₂ isCF₃ and R^(Y1) is methyl.

VIII) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃ and R₄ are as defined in classes and subclasses herein; Y₂and R^(Y1) are independently hydrogen or lower alkyl; R^(8B) is hydrogenor lower alkyl; and e is hydrogen, halogen, —OR^(Y1) or —NR^(Y1)NR^(Y2);wherein R^(Y1) and R² are independently hydrogen, alkyl, heteroalkyl,aryl, heteroaryl or acyl, or R^(Y1) and R^(Y2), taken together with thenitrogen atom to which they are attached, for a heterocyclic orheteroaryl moiety. In certain embodiments, R₃ is hydrogen, lower alkylor an oxygen protecting group. In certain exemplary embodiments, R₃ ismethyl. In certain embodiments, R₄ is halogen, hydroxyl, lower alkoxy,acyloxy or NR⁴AR^(4B), wherein R^(4A) and R^(4B) are independentlyhydrogen, lower alkyl, aryl, acyl or a nitrogen protecting group, orR^(4A) and R^(4B), taken together with the nitrogen atom to which theyare attached, form a substituted or unsusbstituted heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain other embodiments, R^(Y) is OH, NH₂ or halogen (e.g., F). Incertain other embodiments, R^(8B) is hydrogen, methyl or ethyl. Incertain exemplary embodiments, Y₂ is hydrogen or lower alkyl substitutedwith one or more halogen atoms selected from F, Cl, Br and I. In certainexemplary embodiments, Y₂ is hydrogen or methyl substituted with one ormore halogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or CF₃. In certain exemplary embodiments,R^(Y1) is hydrogen or lower alkyl. In certain exemplary embodiments,R^(Y1) is hydrogen or methyl. In certain exemplary embodiments, Y₂ isCF₃ and R^(Y1) is methyl.

IX) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ and n are as defined in classes and subclasses herein; Y₂and R^(Y1) are independently hydrogen or lower alkyl; R₇ is asubstituted or unsubstituted, linear or branched, cyclic or acycliclower alkyl moiety; and R^(8B) is hydrogen or lower alkyl. In certainembodiments, R₃ is hydrogen, lower alkyl or an oxygen protecting group.In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, n is 3. In certain embodiments, R₄ is halogen, hydroxyl,lower alkoxy, acyloxy or NR^(4A)R^(4B), wherein R^(4A) and R^(4B) areindependently hydrogen, lower alkyl, aryl, acyl or a nitrogen protectinggroup, or R^(4A) and R^(4B), taken together with the nitrogen atom towhich they are attached, form a substituted or unsusbstitutedheterocyclic or heteroaryl moiety; or R₄, taken together with the carbonatom to which it is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain other embodiments, R₇ is methyl. In certain otherembodiments, R^(8B) is hydrogen, methyl or ethyl. In certain exemplaryembodiments, Y₂ is hydrogen or lower alkyl substituted with one or morehalogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or methyl substituted with one or morehalogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or CF₃. In certain exemplary embodiments,R^(Y1) is hydrogen or lower alkyl. In certain exemplary embodiments,R^(Y1) is hydrogen or methyl. In certain exemplary embodiments, Y₂ isCF₃ and R^(Y1) is methyl.

X) Compounds of the Formula (and Pharmaceutically Acceptable DerivativesThereof):

wherein R₃-R₆ are as defined in classes and subclasses herein; Y₂ andR^(Y1) are independently hydrogen or lower alkyl; R₇ is a substituted orunsubstituted, linear or branched, cyclic or acyclic lower alkyl moiety;and R^(8B) is hydrogen or lower alkyl. In certain embodiments, R₃ ishydrogen, lower alkyl or an oxygen protecting group. In certainexemplary embodiments, R₃ is methyl. In certain other embodiments, R₅and R₆ are independently lower alkyl. In certain exemplary embodiments,R₅ and R₆ are each methyl. In certain embodiments, R₄ is halogen,hydroxyl, lower alkoxy, acyloxy or NR^(4A)R^(4B), wherein R^(4A) andR^(4B) are independently hydrogen, lower alkyl, aryl, acyl or a nitrogenprotecting group, or R^(4A) and R^(4B), taken together with the nitrogenatom to which they are attached, form a substituted or unsusbstitutedheterocyclic or heteroaryl moiety; or R₄, taken together with the carbonatom to which it is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain other embodiments, R₇ is methyl. In certain otherembodiments, R^(8B) is hydrogen, methyl or ethyl. In certain exemplaryembodiments, Y₂ is hydrogen or lower alkyl substituted with one or morehalogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or methyl substituted with one or morehalogen atoms selected from F, Cl, Br and I. In certain exemplaryembodiments, Y₂ is hydrogen or CF₃. In certain exemplary embodiments,R^(Y1) is hydrogen or lower alkyl. In certain exemplary embodiments,R^(Y1) is hydrogen or methyl. In certain exemplary embodiments, Y₂ isCF₃ and R^(Y1) is methyl.

XI) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ and n are as defined in classes and subclasses herein; andY₂ and R^(Y1) are independently hydrogen or lower alkyl. In certainembodiments, R₃ is hydrogen, lower alkyl or an oxygen protecting group.In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, n is 3. In certain embodiments, R₄ is halogen, hydroxyl,lower alkoxy, acyloxy or NR⁴R^(4B), wherein R^(4A) and R^(4B) areindependently hydrogen, lower alkyl, aryl, acyl or a nitrogen protectinggroup, or R^(4A) and R^(4B), taken together with the nitrogen atom towhich they are attached, form a substituted or unsusbstitutedheterocyclic or heteroaryl moiety; or R₄, taken together with the carbonatom to which it is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain exemplary embodiments, Y₂ is hydrogen or lower alkylsubstituted with one or more halogen atoms selected from F, Cl, Br andI. In certain exemplary embodiments, Y₂ is hydrogen or methylsubstituted with one or more halogen atoms selected from F, Cl, Br andI. In certain exemplary embodiments, Y₂ is hydrogen or CF₃. In certainexemplary embodiments, R^(Y1) is hydrogen or lower alkyl. In certainexemplary embodiments, R^(Y1) is hydrogen or methyl. In certainexemplary embodiments, Y₂ is CF₃ and R^(Y1) is methyl.

XII) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ are as defined in classes and subclasses herein; and Y₂and R^(Y1) are independently hydrogen or lower alkyl. In certainembodiments, R₃ is hydrogen, lower alkyl or an oxygen protecting group.In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, R₄ is halogen, hydroxyl, lower alkoxy, acyloxy orNR^(4A)R^(4B), wherein R^(4A) and R^(4B) are independently hydrogen,lower alkyl, aryl, acyl or a nitrogen protecting group, or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a substituted or unsusbstituted heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain exemplary embodiments, Y₂ is hydrogen or lower alkylsubstituted with one or more halogen atoms selected from F, Cl, Br andI. In certain exemplary embodiments, Y₂ is hydrogen or methylsubstituted with one or more halogen atoms selected from F, Cl, Br andI. In certain exemplary embodiments, Y₂ is hydrogen or CF₃. In certainexemplary embodiments, R^(Y1) is hydrogen or lower alkyl. In certainexemplary embodiments, R^(Y1) is hydrogen or methyl. In certainexemplary embodiments, Y₂ is CF₃ and R^(Y1) is methyl.

XIII) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ and n are as defined in classes and subclasses herein; andY₂ and R^(Y1) are independently hydrogen or lower alkyl. In certainembodiments, R₃ is hydrogen, lower alkyl or an oxygen protecting group.In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, n is 3. In certain embodiments, R₄ is halogen, hydroxyl,lower alkoxy, acyloxy or NR^(4A)R^(4B), wherein R^(4A) and R^(4B) areindependently hydrogen, lower alkyl, aryl, acyl or a nitrogen protectinggroup, or R^(4A) and R^(4B), taken together with the nitrogen atom towhich they are attached, form a substituted or unsusbstitutedheterocyclic or heteroaryl moiety; or R₄, taken together with the carbonatom to which it is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain exemplary embodiments, Y₂ is hydrogen or lower alkylsubstituted with one or more halogen atoms selected from F, Cl, Br andI. In certain exemplary embodiments, Y₂ is hydrogen or methylsubstituted with one or more halogen atoms selected from F, Cl, Br andI. In certain exemplary embodiments, Y₂ is hydrogen or CF₃. In certainexemplary embodiments, R^(Y1) is hydrogen or lower alkyl. In certainexemplary embodiments, R^(Y1) is hydrogen or methyl. In certainexemplary embodiments, Y₂ is CF₃ and R^(Y1) is methyl.

XIV) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ are as defined in classes and subclasses herein; and Y₂and R^(Y1) are independently hydrogen or lower alkyl. In certainembodiments, R₃ is hydrogen, lower alkyl or an oxygen protecting group.In certain exemplary embodiments, R₃ is methyl. In certain otherembodiments, R₅ and R₆ are independently lower alkyl. In certainexemplary embodiments, R₅ and R₆ are each methyl. In certainembodiments, R₄ is halogen, hydroxyl, lower alkoxy, acyloxy orNR^(4A)R^(4B), wherein R^(4A) and R^(4B) are independently hydrogen,lower alkyl, aryl, acyl or a nitrogen protecting group, or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a substituted or unsusbstituted heterocyclic orheteroaryl moiety; or R₄, taken together with the carbon atom to whichit is attached forms a moiety having the structure:

In certain embodiments, R₄ is a halogen selected from fluorine,chlorine, bromine and iodine. In certain exemplary embodiments, R₄ isfluorine. In certain other embodiments, R₄ is F, OH, OAc, NH₂ or R₄,taken together with the carbon atom to which it is attached forms amoiety having the structure:

In certain exemplary embodiments, Y₂ is hydrogen or lower alkylsubstituted with one or more halogen atoms selected from F, Cl, Br andI. In certain exemplary embodiments, Y₂ is hydrogen or methylsubstituted with one or more halogen atoms selected from F, Cl, Br andI. In certain exemplary embodiments, Y₂ is hydrogen or CF₃. In certainexemplary embodiments, R^(Y1) is hydrogen or lower alkyl. In certainexemplary embodiments, R^(Y1) is hydrogen or methyl. In certainexemplary embodiments, Y₂ is CF₃ and R^(Y1) is methyl.

XV) Compounds of the Formula (and Pharmaceutically AcceptableDerivatives Thereof):

wherein R₃-R₆ are as defined in classes and subclasses herein; X₁ is O,NH or CH₂; and Y₁ and Y₂ are independently OH, C(R^(Y1))₃ or Y₁ and Y₂taken together with the carbon atom to which they are attached are —C═O;wherein R^(Y1) is halo. In certain embodiments, R₆ is H or lower alkyl.In certain other embodiments, R₅ is H or lower alkyl. In yet otherembodiments, R₄ is OH. In other embodiments, R₃ is alkyl. In certainexemplary embodiments, X₁ is CH₂, NH or O; Y₁ and Y₂ are independentlyOH, C(R^(Y1))₃ or Y₁ and Y₂ taken together with the carbon atom to whichthey are attached are —C═O, wherein R^(Y1) is halo; R₆ is H or loweralkyl; R₅ is H or lower alkyl; R₄ is OH; and R₃ is alkyl.

It will also be appreciated that for each of the subgroups I-XVdescribed above, a variety of other subclasses are of special interest,including, but not limited to those classes described above i)-cxv) andclasses, subclasses and species of compounds described above and in theexamples herein.

Some of the foregoing compounds can comprise one or more asymmetriccenters, and thus can exist in various isomeric forms, e.g.,stereoisomers and/or diastereomers. Thus, inventive compounds andpharmaceutical compositions thereof may be in the form of an individualenantiomer, diastereomer or geometric isomer, or may be in the form of amixture of stereoisomers. In certain embodiments, the compounds of theinvention are enantiopure compounds. In certain other embodiments,mixtures of stereoisomers or diastereomers are provided.

Furthermore, certain compounds, as described herein may have one or moredouble bonds that can exist as either the Z or E isomer, unlessotherwise indicated. The invention additionally encompasses thecompounds as individual isomers substantially free of other isomers andalternatively, as mixtures of various isomers, e.g., racemic mixtures ofstereoisomers. In addition to the above-mentioned compounds per se, thisinvention also encompasses pharmaceutically acceptable derivatives ofthese compounds and compositions comprising one or more compounds of theinvention and one or more pharmaceutically acceptable excipients oradditives.

Compounds of the invention may be prepared by crystallization ofcompound of formula (I) under different conditions and may exist as oneor a combination of polymorphs of compound of general formula (I)forming part of this invention. For example, different polymorphs may beidentified and/or prepared using different solvents, or differentmixtures of solvents for recrystallization; by performingcrystallizations at different temperatures; or by using various modes ofcooling, ranging from very fast to very slow cooling duringcrystallizations. Polymorphs may also be obtained by heating or meltingthe compound followed by gradual or fast cooling. The presence ofpolymorphs may be determined by solid probe NMR spectroscopy, IRspectroscopy, differential scanning calorimetry, powder X-raydiffractogram and/or other techniques. Thus, the present inventionencompasses inventive compounds, their derivatives, their tautomericforms, their stereoisomers, their polymorphs, their pharmaceuticallyacceptable salts their pharmaceutically acceptable solvates andpharmaceutically acceptable compositions containing them.

As discussed above, this invention provides novel compounds with a rangeof biological properties. Preferred compounds of this invention havebiological activities relevant for the treatment of cancer andangiogenesis-related disorders.

Compounds of this invention include those specifically set forth aboveand described herein, and are illustrated in part by the variousclasses, subgenera and species disclosed elsewhere herein.

Additionally, the present invention provides pharmaceutically acceptablederivatives of the inventive compounds, and methods of treating asubject using these compounds, pharmaceutical compositions thereof, oreither of these in combination with one or more additional therapeuticagents. Certain compounds of the present invention are described in moredetail below. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 75^(th) Ed., inside cover,and specific functional groups are generally defined as describedtherein. Additionally, general principles of organic chemistry, as wellas specific functional moieties and reactivity, are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, the entire contents of which are incorporated herein byreference. Furthermore, it will be appreciated by one of ordinary skillin the art that the synthetic methods, as described herein, utilize avariety of protecting groups. It will be appreciated that the compounds,as described herein, may be substituted with any number of substituentsor functional moieties. In general, the term “substituted” whetherpreceded by the term “optionally” or not, and substituents contained informulas of this invention, refer to the replacement of hydrogenradicals in a given structure with the radical of a specifiedsubstituent. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. As used herein, the term “substituted” is contemplated toinclude all permissible substituents of organic compounds. In a broadaspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnonaromatic substituents of organic compounds. For purposes of thisinvention, heteroatoms such as nitrogen may have hydrogen substituentsand/or any permissible substituents of organic compounds describedherein which satisfy the valencies of the heteroatoms. Furthermore, thisinvention is not intended to be limited in any manner by the permissiblesubstituents of organic compounds. Combinations of substituents andvariables envisioned by this invention are preferably those that resultin the formation of stable compounds useful in the treatment, forexample of proliferative disorders, including, but not limited tocancer. The term “stable”, as used herein, preferably refers tocompounds which possess stability sufficient to allow manufacture andwhich maintain the integrity of the compound for a sufficient period oftime to be detected and preferably for a sufficient period of time to beuseful for the purposes detailed herein.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or a prodrug or other adduct or derivative of a compoundof this invention which upon administration to a patient in need iscapable of providing, directly or indirectly, a compound as otherwisedescribed herein, or a metabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts of amines, carboxylic acids, and other types ofcompounds, are well known in the art. For example, S. M. Berge, et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein byreference. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention, or separately byreacting a free base or free acid function with a suitable reagent, asdescribed generally below. For example, a free base function can bereacted with a suitable acid. Furthermore, where the compounds of theinvention carry an acidic moiety, suitable pharmaceutically acceptablesalts thereof may, include metal salts such as alkali metal salts, e.g.sodium or potassium salts; and alkaline earth metal salts, e.g. calciumor magnesium salts. Examples of pharmaceutically acceptable, nontoxicacid addition salts are salts of an amino group formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid or with organic acids such as aceticacid, oxalic acid, maleic acid, tartaric acid, citric acid, succinicacid or malonic acid or by using other methods used in the art such asion exchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hernisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters that hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moeity advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the compounds of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the issues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” refers tocompounds that are rapidly transformed in vivo to yield the parentcompound of the above formula, for example by hydrolysis in blood. Athorough discussion is provided in T. Higuchi and V. Stella, Pro-drugsas Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, andin Edward B. Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated herein by reference.

2) Synthetic Methodology

In another aspect, the present invention provides methods for preparingnovel macrocycles having formula (I) a described above and in certainclasses and subclasses herein. An overview of exemplary synthesicapproaches to the inventive compounds is provided below, as detailed inSchemes 1-15, and in the Exemplification herein. It will be appreciatedthat the methods as described herein can be applied to each of thecompounds as disclosed herein and equivalents thereof. Additionally, thereagents and starting materials are well known to those skilled in theart. Although the following schemes describe certain exemplarycompounds, it will be appreciated that the use of alternate startingmaterials will yield other analogs of the invention. For example,compounds are described below where X is O; however, it will beappreciated that alternate starting materials and/or intermediates canbe utilized to generate compounds where X is NH, N-alkyl, S, CH₂, etc.

In certain embodiments, compounds as provided herein, for example thosewhere n is 3, X is O, and R₁ and R₂ are each hydrogen, are prepared fromassembly of three segments, as depicted in Scheme 1A below:

In certain other embodiments, compounds as provided herein, for examplethose where n is 3, X is O, R₁ and R₂ are each hydrogen, and Q is amoiety having the structure

are prepared from assembly of five segments, as depicted in Scheme 1Bbelow:

In certain embodiments, compounds of the invention where Q is acarbonyl-containing moiety having the structure:

are prepared from assembly of three segments, as depicted in Scheme 2below:

In certain embodiments, compounds where -Alk-R₈ represents aglutarimide-containing side chain, having the structure:

wherein X, Y, Z and R are as defined in classes and subclasses herein;

are prepared from assembly of three segments, as depicted in Scheme 3below:

wherein G represents a group suitable for effecting theHorner-Wadsworth-Emmons-type coupling.

In certain embodiments, the preparation of fragment A may beaccomplished as depicted in Scheme 4 below:

For example, reduction of commercially available dimethyl2,3-O-isopropylidene-L-tartrate i, followed by diastereoselectivedivinylzinc addition to the in situ generated dialdehyde produces thedesired vinyl carbinol ii (see, Jorgensen et al., J. Org. Chem., 2001,66, 4630). Alkylation (or arylation) of the two hydroxyl groups andremoval of the acetonide protecting group yields diol iii. Glycolcleavage of iii affords α-alkoxy-β-vinyl aldehyde iv. Subjecting iv to aLewis acid catalyzed diene aldehyde condensation (LACDAC) sequence withthe synergistically activated diene v in the presence of TiCl₄, yieldsthe α-chelation controlled dihydropyrone vi (for chelation-controlledcyclocondensations of α-alkoxy aldehydes with synergistically activateddienes, see: Danishefsky et al., J. Am. Chem. Soc., 1985, 107, 1256).The cyclocondensation allows the construction of the three contiguousstereocenters of the macrolide and sets the stage for establishing thetrisubstituted (Z)-alkene C11-C12. Luche reduction of enone vi affordsthe corresponding allylic alcohol, which can be made to undergo anaqueous Ferrier rearrangement to give alcohol vii (for a reference onthe Luche reduction, see: Luche et al., J. Am. Chem. Soc., 1979, 101,5848; for a reference on the Ferrier rearrangement, see: Ferrier, J.Chem. Soc., 1964, 5443). Reductive opening of lactol vii, protection ofthe secobdary hydroxyl group, and oxidation of the primary alcoholyields the C7-C13 core fragment A.

One of ordinary skill in the art will recognize that the protectedhydroxyl (OPG) may be converted to a variety of functional groups,including, but not limited to OH, NH₂ and F, thus allowing access tocompounds where R₄ is OH, OAc, NH₂, F, or R₄, taken together with thecarbon atom to which it is attached forms a moiety having the structure:

among others.

In certain embodiments, coupling of fragment A with a glutarimide moietymay be accomplished as exemplified in Scheme 5 below:

For example, Addition of x to fragment A in the presence of MgCl₂ andTMSCl produces alcohol xi (for a reference reporting a suitable protocolfor anti-selective aldol coupling, see: Evans et al., J. Am. Chem. Soc.,2002, 124, 392). Protection of the resulting secondary hydroxyl groupand reductive cleavage of the chiral auxiliary affords alcohol xii.Coupling of compound xii with the glutarimide side chain may beeffected, for example, via a Horner-Wadsworth-Emmons reaction. Forexample, the Masamune-Roush variant of the Horner-Wadsworth-Emmonsreaction may be used (see: Blanchette et al., Tet. Lett., 1984, 25,2183). Thus, conversion of xii via an oxidation/nucleophilicaddition/oxidation sequence gives β-ketophosphonate xiii. Treatment ofthe phosphonate with LiCl and DBU in the presence of glutarimidealdehyde xiv results in efficient formation of the desired enone xv.

In certain embodiments, formation of the macrolide ring is effected asshown in Scheme 6 below:

For example, removal of the TES protecting group of enone xv yieldsseco-alcohol xvi. A variety of methods for effecting acylation of xviwith dienoic acid may be utilized. For example, a modified Yamaguchiprocedure may be used to give the metathesis precursor xvii (see,Inanaga et al., Bull. Chem. Soc. Jpn., 1979, 52, 1989; and Song et al.,Org. Lett., 2002, 4, 647). A variety of methods for effectingring-closure metathesis of xvii to the desired (E)-isomer may beutilized. For example, subjecting tetraene xvii to ring-closuremetathesis conditions using the second generation Grubbs catalyst givesthe desired macrocyclic (E)-isomer xviii in high yield (see, Scholl etal., Org. Lett., 1999, 1, 953).

Methods for converting the protected hydroxyl group (OPG) into a varietyof functionalities are known in the art. The practitioner skilled in therelevant art will know how to select reagents and reaction conditions toeffect transformation of the protected hydroxyl group (OPG) into adesired functionality FG. In certain embodiments, FG represents OH, NH₂or halogen (e.g., F).

In certain other embodiments, the conjugate ester group present incompound xviii (i.e., at C₂-C₃) may be reduced to the correspondingsaturated ester xix. The practitioner skilled in the relevant art willknow how to select reagents and reaction conditions to effect thistransformation. For example, the Stryker copper hydride may be used(see, Mahoney et al., J. Am. Chem. Soc., 1988, 110, 291), as depicted inSheme 7 below:

In certain embodiments, in Schemes 5-7 above, —X—Y-Z- represents—CH═CH—(CH₂)_(v)— where v is an integer from 1-4. Thus, compound xvdepicted in scheme 5 may have the following structure (xv^(a)):

In certain embodiments, conjugate reduction of this intermediate may beeffected using the stryker reagent, as shown in scheme 8 below:

In certain other embodiments, where further functionalization at C₁₇ ofthe alkyl-glutarimide side chain of xv^(a) is desired, coupling offragment xii with a glutarimide moiety may be accomplished as shown inScheme 9 below:

For example, ephedrine ester xx may be converted to the correspondingWeinreb amide, which is then transformed into the corresponding methylketone upon treatment with MeMgBr. Aldol reaction of ketone xxi withprotected glutarimide aldehyde xxii yields the formation of theC₁₇-hydroxylated adduct xxiii. The practitioner skilled in the relevantart will know how to select reagents and reaction conditions to effecttransformation of this C-17 hydroxyl group into functionalities ofinterest (e.g., alkoxyl, aryloxy, NH₂ or halogen (e.g., F)).

One of ordinary skill in the art will recognize that the ring closingmetathesis coupling may be effected with fragments where at least one ofR₁ and R₂ is not hydrogen, to introduce functionalization at C₆ and/orC₇, as shown in Scheme 10 below. In addition, metathesis reactionconditions may be adjusted so that the (Z)-isomer is predominantlyformed, rather than the (E)-isomer.

One of ordinary skill in the art will also recognize that the inventivemethods for assembling the macrocyclic structure are not limited by theorder in which the different fragments may be put together. Exemplarysynthetic approaches were described in Schemes 1-10 above, whereby theinventive compounds are prepared by (i) nucleophilic addition of Q onfragment A, followed by (ii) ester bond formation between the A-Q adductwith a suitable dienoic acid and (iii) ring closing ring closure to givethe desired macrocyclic scaffold. Other approaches may be used. Forexample, inventive compounds may be prepared by (i) nucleophilicaddition of Q on fragment A, followed by (ii) cross-metathesis reactionof the A-Q adduct obtained in (i) with a suitable dienoic acid and (iii)macrolactonization (i.e., intramolecular ester bond formation) to givethe desired macrocyclic scaffold (See Scheme 11).

Alternatively, inventive compounds may be prepared by (i) nucleophilicaddition of Q on fragment A, followed by (ii) cross-metathesis reactionof the A-Q adduct obtained in (i) with a suitable enone, (iii) acylationof the adduct obtained in (ii) with a suitable reagent and (iv)intramolecular Horner-Wadsworth-Emmons olefination to give the desiredmacrocyclic scaffold (See Scheme 12).

In certain embodiments, the invention provides methods of preparingcompounds where X₁ is NH. Schemes 1-12 above detail exemplary syntheticapproaches for preparing inventive compounds where X₁ is O. A similarapproach may be used to access compounds where X₁ is NH (i.e.,macrolactams). For example, inventive compounds may be prepared by (i)nucleophilic addition of Q on fragment A, followed by (ii) conversion ofthe resulting alcohol to an amine, (iii) amide bond formation betweenthe A-Q adduct formed in (ii) with a suitable dienoic acid and (iv) ringclosing metathesis to give the desired macrolactam scaffold (Scheme 13).

In certain embodiments, inventive compounds may be prepared by (i)nucleophilic addition of Q on fragment A, followed by (ii) conversion ofthe resulting alcohol to the corresponding amine, (iii) cross-metathesisreaction of the A-Q adduct obtained in (ii) with a suitable dienoic acidand (iv) intramolecular amide bond formation to give the desiredmacrolactam scaffold (See Scheme 14).

Alternatively, inventive compounds may be prepared by (i) nucleophilicaddition of Q on fragment A, followed by (ii) conversion of theresulting alcohol to the corresponding amine, (iii) cross-metathesisreaction of the A-Q adduct obtained in (ii) with a suitable enone, (iv)acylation of the adduct obtained in (iii) with a suitable reagent and(v) intramolecular Horner-Wadsworth-Emmons olefination to give thedesired macrocyclic scaffold (See Scheme 15).

Other approaches to prepare inventive compounds will be readily apparentto the practitioner skilled in the relevant art.

Diversification:

It will also be appreciated that each of the components used in thesynthesis of Migrastatin analogues can be diversified either beforesynthesis or alternatively after the construction of the macrocycle. Asused herein, the term “diversifying” or “diversify” means reacting aninventive compound (I) or any of the precursor fragments (e.g., (A)etc.) as defined herein (or any classes or subclasses thereof) at one ormore reactive sites to modify a functional moiety or to add a functionalmoiety (e.g., nucleophilic addition of a substrate). Described generallyherein are a variety of schemes to assist the reader in the synthesis ofa variety of analogues, either by diversification of the intermediatecomponents or by diversification of the macrocyclic structures asdescribed herein, and classes and subclasses thereof. It will also beappreciated that although many of the schemes herein depict 14-memberedmacrocycles, the reactions described herein may also be applied to otherring structures (for example to 12-, 13- and 15-membered ringstructures). It will be appreciated that a variety of diversificationreactions can be employed to generate novel analogues. As but a fewexamples, epoxidation and aziridation can be conducted to generateepoxide and aziridine analogues of compounds described herein.Additionally, addition across either double bond will generateadditional diversity. In addition to diversification aftermacrocyclization, it will be understood that diversification can occurprior to macrocyclization (e.g., epoxidation, aziridation, reduction ata C₂₋₃ and/or C₁₂₋₁₃ double bond(s) could occur prior to metathesisring-closure, or other means known in the art to effect macorcyclic ringclosure, to describe just one example). For additional guidanceavailable in the art, the practitioner is directed to “Advanced OrganicChemistry”, March, J. John Wiley & Sons, 2001, 5^(th) ed., the entirecontents of which are hereby incorporated by reference.

In certain embodiments, the present invention provides a method forpreparing a Migrastatin analog having the structure:

said method comprising steps of:

-   a. reacting a fragment Q with a compound having the structure:

under suitable conditions to effect formation of an A-Q adduct havingthe structure:

-   b. reacting A-Q formed in step a with a dienoic acid having the    structure:

under suitable conditions to effect formation of an ester having thestructure:

-   c. subjecting the ester formed in step b to ring closing metathesis    reaction conditions to effect formation of the macrolide having the    structure:

wherein R₁ and R₂ are each independently hydrogen, halogen, —CN,—S(O)₁₋₂ R^(1A), NO₂, COR^(1A), —CO₂R^(1A), —NR^(1A)C(═O)R^(1B),—NR^(1A)C(═O)OR^(1B), —CONR^(1A)R^(1B), an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(1A);wherein W is independently —O—, —S— or —NR^(1C)—, wherein eachoccurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R₁ and R₂, taken together, form an alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₃ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group;

R₄ is, —OR^(4A), —OC(═O)R^(4A) or —NR^(4A)R^(4B); wherein R^(4A) andR^(4B) are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; a prodrug moiety,a nitrogen protecting group or an oxygen protecting group; or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a heterocyclic or heteroaryl moiety; or R₄, takentogether with the carbon atom to which it is attached forms a moietyhaving the structure:

R₅ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), NO₂, —COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(6C)—, wherein each occurrence of R^(6A), R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R₆ and R_(c) takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,—CN, —S(O)₁₂R^(a1), —NO₂, —COR^(a1), —CO₂R^(a1), —NR^(a1)C(═O)R^(a2),—NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2), an anlphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(a1);wherein W is independently —O—, —S— or —NR^(a3)—, wherein eachoccurrence of R^(a1), R^(a2) and R^(a3) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —COR^(c1),—CO₂R^(c1), —NR^(c1)C(═O)R^(c2), —NR^(c1)C(═O)OR^(c2), —CONR^(c1)R^(c2);an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(c1); wherein W is independently —O—, —S— or—NR^(c3)—, wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R_(c) and R₆, takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

n is an integer from 1 to 5; and

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; and

pharmaceutically acceptable derivatives thereof.

In certain embodiments, the method further comprises steps ofdiversifying the macrolide obtained in step c to form a Migrastatinanalog with the desired functionalization.

In certain embodiments, the present invention provides a method forpreparing a Migrastatin analog having the structure:

said method comprising steps of:

-   a. reacting a fragment Q with a compound having the structure:

under suitable conditions to effect formation of an A-Q adduct havingthe structure:

-   b. reacting A-Q formed in step a with a dienoic acid having the    structure:

-   -   under suitable conditions to effect formation of an olefin        having the structure:

-   c. subjecting the olefin formed in step b to suitable conditions to    effect formation of the macrolide having the structure:

wherein R₁ and R₂ are each independently hydrogen, halogen, —CN,—S(O)₁₋₂R^(1A), —NO₂, COR^(1A), —CO₂R^(1A), —NR^(1A)C(═O)R^(1B),—NR^(1A)C(═O)OR^(1B), —CONR^(1A)R^(1B), an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(1A);wherein W is independently —O—, —S— or —NR^(1C)—, wherein eachoccurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R₁ and R₂, taken together with the carbon atoms towhich they are attached, form an alicyclic, heteroalicyclic, aryl orheteroaryl moiety;

R₃ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group;

R₄ is halogen, —O^(4A), —OC(═O)R^(4A) or —NR^(4A)R^(4B); wherein R^(4A)and R^(4B) are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; a prodrug moiety,a nitrogen protecting group or an oxygen protecting group; or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a heterocyclic or heteroaryl moiety; or R₄, takentogether with the carbon atom to which it is attached forms a moietyhaving the structure:

R₅ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), NO₂, —COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(6C)—, wherein each occurrence of R^(6A), R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R₆ and R_(c), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,—CN, —S(O)₁₋₂R^(a1), —NO₂, —COR^(a1), —CO₂R^(a1), —NR^(a1)C(═O)R^(a2),—NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2), an alphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(a1);wherein W is independently —O—, —S— or —NR^(a3)—, wherein eachoccurrence of R^(a1), R^(a2) and R^(a3) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —COR^(c1),—CO₂R^(c1), —NR^(c1)C(═O)R^(c2), —NR^(c1)C(═)OR^(c2), —CONR^(c1); analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(c1); wherein W is independently —O—, —S— or—NR^(c3)—, wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R_(c) and R₆, takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

n is an integer from 1 to 5; and

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; and

pharmaceutically acceptable derivatives thereof.

In certain embodiments, the method further comprises steps ofdiversifying the macrolide obtained in step c to form a Migrastatinanalog with the desired functionalization.

In certain embodiments, the present invention provides a method forpreparing a Migrastatin analog having the structure:

said method comprising steps of:

-   a. reacting a fragment Q with a compound having the structure:

under suitable conditions to effect formation of an A-Q adduct havingthe structure:

-   b. reacting A-Q formed in step a with n enone having the structure:

-   -   under suitable conditions to effect formation of an olefin        having the structure:

-   c. acylating the olefin formed in step b with a suitable reagent    under suitable conditions to effect formation of an intermediate    having the structure:

wherein G is a group suitable to effect ring closure; and

-   d. subjecting the intermediate formed in step c to suitable    conditions to effect formation of the macrolide having the    structure:

wherein R₁ and R₂ are each independently hydrogen, halogen, —CN,—S(O)₁₋₂R^(1A), NO₂, COR^(1A), —CO₂R^(1A),—NR^(1A)C(═O)R^(1B)—NR^(1A)C(═O)OR^(1B)—CONR^(1A)R^(1B), an aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aryl or heteroaryl moiety,or —WR^(1A); wherein W is independently —O—, —S— or —NR^(1C)—, whereineach occurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen,or an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R₁ and R₂, taken together with the carbon atoms towhich they are attached, form an alicyclic, heteroalicyclic, aryl orheteroaryl moiety;

R₃ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group; R₄ is halogen, —OR^(4A), —OC(═O)R^(4A) or—NR^(4A)R^(4B); wherein R^(4A) and R^(4B) are independently hydrogen, analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; a prodrug moiety, a nitrogen protecting group or anoxygen protecting group; or R^(4A) and R^(4B), taken together with thenitrogen atom to which they are attached, form a heterocyclic orheteroaryl moiety;

R₅ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), —NO₂, COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(6C)—, wherein each occurrence of R^(6A)R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R₆ and R_(c), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,—CN, —S(O)₁₂R^(a1), —NO₂, —COR^(a1), —CO₂R^(a1), —NR^(a1)C(═O)R^(a2),—NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2), an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(a1);wherein W is independently —O—, —S— or —NR^(a3)—, wherein eachoccurrence of R^(a1), R^(a2) and R^(a3) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —COR^(c1),—CO₂R^(c1), —NR^(c1)C(═O)R^(c2), NR^(c1)CC(═O)OR^(a), —CONR^(c2)R^(c2);an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(c1); wherein W is independently —O—, —S— or—NR^(c3)—, wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R_(c) and R₆, takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

n is an integer from 1 to 5; and

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; and

pharmaceutically acceptable derivatives thereof.

In certain embodiments, G is —P(═O)R′₂ and step d involves subjectingthe intermediate formed in step c to Horner-Wadsworth-Emmons reactionconditions to effect formation of the macrolide. In certain otherembodiments, the method further comprises steps of diversifying themacrolide obtained in step d to form a Migrastatin analog with thedesired functionalization.

In certain embodiments, the present invention provides a method forpreparing a Migrastatin analog having the structure:

said method comprising steps of:

-   a. reacting a fragment Q with a compound having the structure:

-   -   under suitable conditions to effect formation of an alcohol        adduct having the structure:

-   b. converting the alcohol adduct formed in step a under suitable    conditions to form an amine having the structure:

c. reacting the amine formed in step b with a dienoic acid having thestructure:

-   -   under suitable conditions to effect formation of an amide having        the structure:

-   d. subjecting the amide formed in step c to ring closing metathesis    reaction conditions to effect formation of the macrolide having the    structure:

wherein R₁ and R₂ are each independently hydrogen, halogen, —CN,—S(O)₁₋₂R^(1A), NO₂, COR^(1A), —CO₂R^(1A), —NR^(1A)C(═O)R^(1B),—NR^(1A)C(═O)OR^(1B)—CONR^(1A)R^(1B), an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(1A);wherein W is independently —O—, —S— or —NR^(1C)—, wherein eachoccurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R₁ and R₂, taken together with the carbon atoms towhich they are attached, form an alicyclic, heteroalicyclic, aryl orheteroaryl moiety;

R₃ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group;

R₄ is halogen, —OR^(4A), —OC(═O)R^(4A) or —NR^(4A)R^(4B); wherein R^(4A)and R^(4B) are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; a prodrug moiety,a nitrogen protecting group or an oxygen protecting group; or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a heterocyclic or heteroaryl moiety;

R₅ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), —NO₂, COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(6C)—, wherein each occurrence of R^(6A)R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R₆ and R_(c), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,—CN, —S(O)₁₂R^(a1), —NO₂, —COR^(a1), —CO₂R^(a1), —NR^(a1)C(═O)R^(a2),—NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2), an alphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(a1);wherein W is independently —O—, —S— or —NR^(a3)—, wherein eachoccurrence of R^(a1)—, R^(a2) and R^(a3) is independently hydrogen, oran aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —COR^(c1),—CO₂R^(c1), —NR^(c1)C(═O)R^(c2), NR^(c1)C(═O)OR^(c2), —CONR^(c1)R^(c2);an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR¹; wherein W is independently —O—, —S— or—NR^(c3)—, wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R_(c) and R₆, takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

n is an integer from 1 to 5;

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; and pharmaceuticallyacceptable derivatives thereof.

In certain embodiments, the method further comprises steps ofdiversifying the macrolide obtained in step d to form a macrolactam(i.e., Migrastatin analog) with the desired functionalization.

In certain embodiments, the present invention provides a method forpreparing a Migrastatin analog having the structure:

said method comprising steps of:

-   b. reacting a fragment Q with a compound having the structure:

-   -   under suitable conditions to effect formation of an alcohol        adduct having the structure:

b. converting the alcohol adduct formed in step a under suitableconditions to form an amine having the structure:

-   d. reacting the amine formed in step b with a dienoic acid having    the structure:

-   -   under suitable conditions to effect formation of an olefin        having the structure:

-   e. subjecting the olefin formed in step c to suitable conditions to    effect formation of the macrolactam having the structure:

wherein R₁ and R₂ are each independently hydrogen, halogen, —CN,—S(O)₁₋₂R^(1A), —NO₂, COR^(1A), —CO₂R^(1A), NR^(1A)C(═O)R^(1B),—NR^(1A)C(═O)OR^(1B), —CONR^(1A)R^(1B), an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(1A);wherein W is independently —O—, —S— or —NR^(1C)—, wherein eachoccurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R₁ and R₂, taken together with the carbon atoms towhich they are attached, form an alicyclic, heteroalicyclic, aryl orheteroaryl moiety;

R₃ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group;

R₄ is halogen, —OR^(4A), —OC(═O)_(R) ^(4A) or —NR^(4A)R^(4B); whereinR^(4A) and R^(4B) are independently hydrogen, an aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aryl or heteroaryl moiety;a prodrug moiety, a nitrogen protecting group or an oxygen protectinggroup; or R^(4A) and R^(4B), taken together with the nitrogen atom towhich they are attached, form a heterocyclic or heteroaryl moiety;

R₅ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), —NO₂, —COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B)an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(6C)—, wherein each occurrence of R^(6A)R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R₆ and R_(c), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,—CN, —S(O)₁₂R^(a1), —NO₂, —COR^(a1), —CO₂R^(a1), —NR^(a1)C(═O)R^(a2),—NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2), an alphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(a1);wherein W is independently —O—, —S— or —NR^(a3)—, wherein eachoccurrence of R^(a1), R^(a2) and R^(a3) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —CO₂R^(c1),—NR^(c1)C(═O)R^(c2); —NR^(c1)C(═O)OR^(c2), —CONR^(c1)R^(c2); analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(c1); wherein W is independently —O—, —S— or—NR^(c3)—, wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R_(c) and R₆, takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

n is an integer from 1 to 5;

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; and

pharmaceutically acceptable derivatives thereof.

In certain embodiments, the method further comprises steps ofdiversifying the macrolide obtained in step e to form a macrolactam(i.e., Migrastatin analog) with the desired functionalization.

In certain embodiments, the present invention provides a method forpreparing a Migrastatin analog having the structure:

said method comprising steps of:

-   a. reacting a fragment Q with a compound having the structure:

under suitable conditions to effect formation of an A-Q adduct havingthe structure:

-   b. converting the alcohol adduct formed in step a under suitable    conditions to form an amine having the structure:

-   c. reacting the amine formed in step b with an enone having the    structure:

-   -   under suitable conditions to effect formation of an olefin        having the structure:

-   d. acylating the olefin formed in step c with a suitable reagent    under suitable conditions to effect formation of an intermediate    having the structure:

wherein G is a group suitable to effect ring closure; and

-   e. subjecting the intermediate formed in step d to suitable    conditions to effect formation of the macrolide having the    structure:

wherein R₁ and R₂ are each independently hydrogen, halogen, —CN,—S(O)₁₋₂R^(1A), NO₂, COR^(1A), —CO₂R^(1A), —NR^(1A)C(═O)R^(1B),—NR^(1A)C(═O)OR^(1B), —CONR^(1A)R^(1B), an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(1A);wherein W is independently —O—, —S— or —NR^(1C)—, wherein eachoccurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R₁ and R₂, taken together with the carbon atoms towhich they are attached, form an alicyclic, heteroalicyclic, aryl orheteroaryl moiety;

R₃ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group;

R₄ is halogen, —OR^(4A), —OC(═O)R^(4A) or —NR^(4A)R^(4B); wherein R^(4A)and R^(4B) are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; a prodrug moiety,a nitrogen protecting group or an oxygen protecting group; or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a heterocyclic or heteroaryl moiety;

R₅ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), —NO₂, COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B)an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(6C)—, wherein each occurrence of R^(6A)R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R₆ and R_(c), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,—CN, —S(O)₁₂R^(a1), —NO₂, —COR^(a1), —CO₂R^(a1), —NR^(a1)C(═O)R^(a2),—NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2), an alphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(a1);wherein W is independently —O—, —S— or —NR^(a3)—, wherein eachoccurrence of R^(a1), R^(a2) and R^(a3) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —COR^(c1),—CO₂R^(c1), —NR^(c1)C(═O)R^(c2), —NR^(c1)C(═O)OR^(c2), —CONR^(c1)R^(c2);an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(c1); wherein W is independently —O—, —S— or—NR^(c3)—, wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R_(c) and R₆, takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

n is an integer from 1 to 5; and

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(A1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; and

pharmaceutically acceptable derivatives thereof.

In certain embodiments, G is —P(═O)R′₂ and step e involves subjectingthe intermediate formed in step d to Horner-Wadsworth-Emmons reactionconditions to effect formation of the macrolide. In certain otherembodiments, the method further comprises steps of diversifying themacrolide obtained in step e to form a Migrastatin analog with thedesired functionalization.

3) Pharmaceutical Compositions

Iin another aspect of the present invention, pharmaceutical compositionsare provided, which comprise any one of the compounds described herein(or a prodrug, pharmaceutically acceptable salt or otherpharmaceutically acceptable derivative thereof), and optionally comprisea pharmaceutically acceptable carrier, adjuvant or vehicle. In certainother embodiments, the compositions of the invention are useful for thetreatment of cancer and disorders associated with metastasis and/orangiogenesis. In certain embodiments, the inventive compositionsoptionally further comprise one or more additional therapeutic agents.In certain other embodiments, the additional therapeutic agent is acytotoxic agent, as discussed in more detail herein. In certain otherembodiments, the additional therapeutic agent is an anticancer agent. Incertain embodiments, the anticancer agent is an epothilone, taxol,radicicol or TMC-95A/B. In certain embodiments, the epothilone is12,13-desoxyepothilone B, (E)-9,10-dehydro-12,13-desoxyEpoB and26-CF3-(E)-9,10-dehydro-12,13-desoxyEpoB. Alternatively, a compound ofthis invention may be administered to a patient in need thereof incombination with the administration of one or more other therapeuticagents. For example, additional therapeutic agents for conjointadministration or inclusion in a pharmaceutical composition with acompound of this invention may be an antiangiogenesis agent oranticancer agent approved for the treatment of cancer, as discussed inmore detail herein, or it may be any one of a number of agentsundergoing approval in the Food and Drug Administration that ultimatelyobtain approval for the treatment of cancer.

As described above, the pharmaceutical compositions of the presentinvention additionally comprise a pharmaceutically acceptable carrier,adjuvant or vehicle, which, as used herein, includes any and allsolvents, diluents, or other liquid vehicle, dispersion or suspensionaids, surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. Remington's PharmaceuticalSciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton,Pa., 1980) discloses various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional carrier medium is incompatible with thecompounds of the invention, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatine; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; cornoil and soybean oil; glycols; such as propylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogenfree water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension orcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionthat, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude (poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar—agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose and starch. Such dosage forms may alsocomprise, as in normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such asmagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions,which can be used, include polymeric substances and waxes.

The present invention encompasses pharmaceutically acceptable topicalformulations of inventive compounds. The term “pharmaceuticallyacceptable topical formulation”, as used herein, means any formulationwhich is pharmaceutically acceptable for intradermal administration of acompound of the invention by application of the formulation to theepidermis. In certain embodiments of the invention, the topicalformulation comprises a carrier system. Pharmaceutically effectivecarriers include, but are not limited to, solvents (e.g., alcohols, polyalcohols, water), creams, lotions, ointments, oils, plasters, liposomes,powders, emulsions, microemulsions, and buffered solutions (e.g.,hypotonic or buffered saline) or any other carrier known in the art fortopically administering pharmaceuticals. A more complete listing ofart-known carriers is provided by reference texts that are standard inthe art, for example, Remington's Pharmaceutical Sciences, 16th Edition,1980 and 17th Edition, 1985, both published by Mack Publishing Company,Easton, Pa., the disclosures of which are incorporated herein byreference in their entireties. In certain other embodiments, the topicalformulations of the invention may comprise excipients. Anypharmaceutically acceptable excipient known in the art may be used toprepare the inventive pharmaceutically acceptable topical formulations.Examples of excipients that can be included in the topical formulationsof the invention include, but are not limited to, preservatives,antioxidants, moisturizers, emollients, buffering agents, solubilizingagents, other penetration agents, skin protectants, surfactants, andpropellants, and/or additional therapeutic agents used in combination tothe inventive compound. Suitable preservatives include, but are notlimited to, alcohols, quaternary amines, organic acids, parabens, andphenols. Suitable antioxidants include, but are not limited to, ascorbicacid and its esters, sodium bisulfite, butylated hydroxytoluene,butylated hydroxyanisole, tocopherols, and chelating agents like EDTAand citric acid. Suitable moisturizers include, but are not limited to,glycerine, sorbitol, polyethylene glycols, urea, and propylene glycol.Suitable buffering agents for use with the invention include, but arenot limited to, citric, hydrochloric, and lactic acid buffers. Suitablesolubilizing agents include, but are not limited to, quaternary ammoniumchlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.Suitable skin protectants that can be used in the topical formulationsof the invention include, but are not limited to, vitamin E oil,allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the pharmaceutically acceptable topicalformulations of the invention comprise at least a compound of theinvention and a penetration enhancing agent. The choice of topicalformulation will depend or several factors, including the condition tobe treated, the physicochemical characteristics of the inventivecompound and other excipients present, their stability in theformulation, available manufacturing equipment, and costs constraints.As used herein the term “penetration enhancing agent” means an agentcapable of transporting a pharmacologically active compound through thestratum corneum and into the epidermis or dermis, preferably, withlittle or no systemic absorption. A wide variety of compounds have beenevaluated as to their effectiveness in enhancing the rate of penetrationof drugs through the skin. See, for example, Percutaneous PenetrationEnhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., BocaRaton, Fla. (1995), which surveys the use and testing of various skinpenetration enhancers, and Buyuktimkin et al., Chemical Means ofTransdermal Drug Permeation Enhancement in Transdermal and Topical DrugDelivery Systems, Gosh T. K., Pfister W. R., Yum S. I. (Eds.),Interpharm Press Inc., Buffalo Grove, Ill. (1997). In certain exemplaryembodiments, penetration agents for use with the invention include, butare not limited to, triglycerides (e.g., soybean oil), aloe compositions(e.g., aloe-vera gel), ethyl alcohol, isopropyl alcohol,octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400,propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g.,isopropyl myristate, methyl laurate, glycerol monooleate, and propyleneglycol monooleate) and N-methylpyrrolidone.

In certain embodiments, the compositions may be in the form ofointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants or patches. In certain exemplary embodiments, formulations ofthe compositions according to the invention are creams, which mayfurther contain saturated or unsaturated fatty acids such as stearicacid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleylalcohols, stearic acid being particularly preferred. Creams of theinvention may also contain a non-ionic surfactant, for example,polyoxy-40-stearate. In certain embodiments, the active component isadmixed under sterile conditions with a pharmaceutically acceptablecarrier, adjuvant or vehicle and any needed preservatives or buffers asmay be required. Ophthalmic formulation, eardrops, and eye drops arealso contemplated as being within the scope of this invention.Additionally, the present invention contemplates the use of transdermalpatches, which have the added advantage of providing controlled deliveryof a compound to the body. Such dosage forms are made by dissolving ordispensing the compound in the proper medium. As discussed above,penetration enhancing agents can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the compound in apolymer matrix or gel.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be formulated and employed incombination therapies, that is, the compounds and pharmaceuticalcompositions can be formulated with or administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, an inventive compound may beadministered concurrently with another anticancer agent), or they mayachieve different effects (e.g., control of any adverse effects).

For example, other therapies or therapeutic agents that may be used incombination with the inventive compounds of the present inventioninclude surgery, radiotherapy (in but a few examples, γ-radiation,neutron beam radiotherapy, electron beam radiotherapy, proton therapy,brachytherapy, and systemic radioactive isotopes, to name a few),endocrine therapy, biologic response modifiers (interferons,interleukins, and tumor necrosis factor (TNF) to name a few),hyperthermia and cryotherapy, agents to attenuate any adverse effects(e.g., antiemetics), and other approved chemotherapeutic drugs,including, but not limited to, alkylating drugs (mechlorethamine,chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites(Methotrexate), purine antagonists and pyrimidine antagonists(6-Mercaptopurine, 5-Fluorouracil, Cytarabile, Gemcitabine), spindlepoisons (Vinblastine, Vincristine, Vinorelbine, Paclitaxel),podophyllotoxins (Etoposide, Irinotecan, Topotecan), antibiotics(Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (Carmustine,Lomustine), inorganic ions (Cisplatin, Carboplatin), enzymes(Asparaginase), and hormones (Tamoxifen, Page 111 of 217 Leuprolide,Flutamide, and Megestrol), to name a few. For a more comprehensivediscussion of updated cancer therapies see, The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference. See also the National Cancer Institute (CNI)website (www.nci.nih.gov) and the Food and Drug Administration (FDA)website for a list of the FDA approved oncology drugs(www.fda.gov/cder/cancer/druglistframe—See Appendix A).

In certain embodiments, the pharmaceutical compositions of the presentinvention further comprise one or more additional therapeutically activeingredients (e.g., chemotherapeutic and/or palliative). For purposes ofthe invention, the term “Palliative” refers to treatment that is focusedon the relief of symptoms of a disease and/or side effects of atherapeutic regimen, but is not curative. For example, palliativetreatment encompasses painkillers, antinausea medications andanti-sickness drugs. In addition, chemotherapy, radiotherapy and surgerycan all be used palliatively (that is, to reduce symptoms without goingfor cure; e.g., for shrinking tumors and reducing pressure, bleeding,pain and other symptoms of cancer).

4) Research Uses, Pharmaceutical Uses and Methods of Treatment

Research Uses

According to the present invention, the inventive compounds may beassayed in any of the available assays known in the art for identifyingcompounds having antiangiogenic activity and/or antiproliferativeactivity. For example, the assay may be cellular or non-cellular, invivo or in vitro, high- or low-throughput format, etc.

Thus, in one aspect, compounds of this invention which are of particularinterest include those which:

-   -   exhibit activity as inhibitors of cell migration;    -   exhibit an antiproliferative and/or an antiangiogenic effect on        solid tumors; and/or    -   exhibit a favorable therapeutic profile (e.g., safety, efficacy,        and stability).

As discussed above, certain of the compounds as described herein exhibitactivity generally as inhibitors of cell migration and/or angiogenesis.More specifically, compounds of the invention act as inhibitors of tumorgrowth and angiogenesis.

As detailed in the exemplification herein, in assays to determine theability of compounds to inhibit tumor cell migration (e.g., chamber cellmigration assay), certain inventive compounds exhibited IC₅₀ values ≦50μM. In certain other embodiments, inventive compounds exhibit IC₅₀values ≦40 μM. In certain other embodiments, inventive compoundsexhibited IC₅₀ values ≦30 μM. In certain other embodiments, inventivecompounds exhibited IC₅₀ values ≦20 μM. In certain other embodiments,inventive compounds exhibited IC₅₀ values ≦10 μM. In certain otherembodiments, inventive compounds exhibited IC₅₀ values ≦7.5 μM. Incertain embodiments, inventive compounds exhibited IC₅₀ values ≦5 μM. Incertain other embodiments, inventive compounds exhibit IC₅₀ values ≦2.5μM. In certain embodiments, inventive compounds exhibited IC₅₀ values ≦1μM. In certain other embodiments, inventive compounds exhibited IC₅₀values ≦750 nM. In certain other embodiments, inventive compoundsexhibited IC₅₀ values ≦500 nM. In certain other embodiments, inventivecompounds exhibited IC₅₀ values ≦250 nM. In certain other embodiments,inventive compounds exhibited IC₅₀ values ≦100 nM. In other embodiments,exemplary compounds exhibited IC₅₀ values ≦75 nM. In other embodiments,exemplary compounds exhibited IC₅₀ values ≦50 nM. In other embodiments,exemplary compounds exhibit IC₅₀ values ≦40 nM. In other embodiments,exemplary compounds exhibited IC₅₀ values ≦30 nM. In other embodiments,exemplary compounds exhibited IC₅₀ values ≦25 nM.

As detailed in the exemplification herein, in assays to determine theability of compounds to inhibit tumor cell proliferation, certaininventive compounds exhibit IC₅₀ values ≦200 μM. In certain otherembodiments, inventive compounds exhibit IC₅₀ values ≦150 μM. In certainother embodiments, inventive compounds exhibit IC₅₀ values ≦100 μM. Incertain other embodiments, inventive compounds exhibit IC₅₀ values ≦50μM. In certain other embodiments, inventive compounds exhibit IC₅₀values ≦10 μM. In certain other embodiments, inventive compounds exhibitIC₅₀ values ≦7.5 μM. In certain embodiments, inventive compounds exhibitIC₅₀ values ≦5 μM. In certain other embodiments, inventive compoundsexhibit IC₅₀ values ≦2.5 μM. In certain embodiments, inventive compoundsexhibit IC₅₀ values ≦1 μM. In certain other embodiments, inventivecompounds exhibit IC₅₀ values ≦750 nM. In certain other embodiments,inventive compounds exhibit IC₅₀ values ≦500 nM. In certain otherembodiments, inventive compounds exhibit IC₅₀ values ≦250 nM. In certainother embodiments, inventive compounds exhibit IC₅₀ values ≦100 nM. Inother embodiments, exemplary compounds exhibit IC₅₀ values ≦75 nM. Inother embodiments, exemplary compounds exhibit IC₅₀ values ≦50 nM.

In certain embodiments, the present invention provides methods foridentifying Migrastatin analogs useful in the preparation ofpharmaceutical compositions for the treatment of various disordersincluding cancer, metastasis and disorders involving increasedangiogenesis.

In certain exemplary embodiments, there is provided a method foridentifying Migrastatin analogs having anti-angiogenic activity, themethod comprising steps of:

-   -   a. contacting a compound with a plurality of cells, whereby the        compound has the structure:

or pharmaceutically acceptable derivative thereof;

wherein R₁ and R₂ are each independently hydrogen, halogen, —CN,—S(O)₁₋₂R^(1A), NO₂, —COR^(1A), —CO₂R^(1A), NR^(1A)C(═O)R^(1B),—NR^(1A)C(═O)OR^(1B), —CONR^(1A)R^(1B), an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(1A);wherein W is independently —O—, —S— or —NR^(1C)—, wherein eachoccurrence of R^(1A), R^(1B) and R^(1C) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R₁ and R₂, taken together with the carbon atoms towhich they are attached, form an alicyclic, heteroalicyclic, aryl orheteroaryl moiety;

R₃ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or a prodrug moiety or anoxygen protecting group;

R₄ is halogen, —OR^(4A), —OC(═O)R^(4A) or —NR^(4A)R^(4B); wherein R^(4A)and R^(4B) are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; a prodrug moiety,a nitrogen protecting group or an oxygen protecting group; or R^(4A) andR^(4B), taken together with the nitrogen atom to which they areattached, form a heterocyclic or heteroaryl moiety;

R₅ is hydrogen, an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

R₆ is hydrogen, halogen, —CN, —S(O)₁₋₂R^(6A), —NO₂, —COR^(6A),—CO₂R^(6A), —NR^(6A)C(═O)R^(6B), —NR^(6A)C(═O)OR^(6B), —CONR^(6A)R^(6B),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(6A); wherein W is independently —O—, —S— or—NR^(6C)—, wherein each occurrence of R^(6A), R^(6B) and R^(6C) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R₆ and R_(c), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(a) and each occurrence of R_(b) are independently hydrogen, halogen,—CN, —S(O)₁₋₂R^(a1), —NO₂, —COR^(a1), —CO₂R^(a1), —NR^(a1)C(═O)R^(a2),—NR^(a1)C(═O)OR^(a2), —CONR^(a1)R^(a2), an alphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or —WR^(a1);wherein W is independently —O—, —S— or —NR^(a3)—, wherein eachoccurrence of R^(a1), R^(a2) and R^(a3) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or R_(a) and the adjacent occurrence of R_(b), takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

R_(c) is hydrogen, halogen, —CN, —S(O)₁₋₂R^(c1), —NO₂, —CO₂R^(c1),—NR^(c1)C(═O)R^(c2), —NR^(c1)C(═O)OR^(c2), —CONR^(c1)R^(c2); analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(c1); wherein W is independently —O—, —S— orNR^(c3) wherein each occurrence of R^(c1), R^(c2) and R^(c3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety; or R_(c) and R₆, takentogether with the carbon atoms to which they are attached, form analicyclic, heteroalicyclic, aryl or heteroaryl moiety;

n is an integer from 1 to 5;

X₁ is O, S, NR^(X1) or CR^(X1)R^(X2); wherein R^(X1) and R^(X2) areindependently hydrogen, halogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety, or a nitrogenprotecting group;

Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1),—CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2),an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety, or —WR^(Q1); wherein W is independently —O—, —S— or—NR^(Q3)—, wherein each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or an aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aryl or heteroaryl moiety;

Y₁ and Y₂ are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; or —WR^(Y1);wherein W is independently —O—, —S— or —NR^(Y2)—, wherein eachoccurrence of R^(Y1) and R^(Y2) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or Y₁ and Y₂ together with the carbon atom to whichthey are attached form a moiety having the structure:

-   -   b. evaluating the effect of the compound on the complexity of        the tube network among the cells.

In certain embodiments, the compound being contacted with the pluralityof cells is at a concentration ≦200 μM. In certain exemplaryembodiments, the compound has the following stereochemistry:

In certain embodiments, in the method described directly above, the stepof evaluating comprises comparing the disturbance of the complexity ofthe tube network with that observed for cells exposed to a referenceMigrastatin concentration. In certain exemplary embodiments, thereference Migrastatin concentration is about 100 μM. In certainexemplary embodiments, the reference Migrastatin concentration is about75 μM. In certain exemplary embodiments, the reference Migrastatinconcentration is about 50 μM. In certain exemplary embodiments, thereference Migrastatin concentration is about 30 μM.

In certain embodiments, the method is for identifying Migrastatinanalogs useful in the preparation of pharmaceutical compositions for thetreatment of angiogenesis-related disorders.

In certain other embodiments, the invention provides a highthroughputmethod for identifying Migrastatin analogs having anti-angiogenicactivity, the method comprising steps of:

-   -   a. introducing in each of a plurality of reaction vessels:

a plurality of cells; and one or more test compounds with having thestructure (I) as defined generally above and in classes and subclassesherein; or pharmaceutically acceptable derivative thereof; and

-   -   b. evaluating in each reaction vessel the effect of the test        compound on the complexity of the tube network in the cells.

In certain embodiments, the test compound being contacted with theplurality of cells is at a concentration ≦200 μM. In certain exemplaryembodiments, the test compound has the following stereochemistry:

In certain embodiments, in the method described directly above, the stepof evaluating comprises comparing the disturbance of the complexity ofthe tube network in each reaction vessel with that observed for cellsexposed to a reference Migrastatin concentration. In certain exemplaryembodiments, the reference Migrastatin concentration is about 100 μM. Incertain exemplary embodiments, the reference Migrastatin concentrationis about 75 μM. In certain exemplary embodiments, the referenceMigrastatin concentration is about 50 μM. In certain exemplaryembodiments, the reference Migrastatin concentration is about 30 μM.

In certain embodiments, the method is for identifying Migrastatinanalogs useful in the preparation of pharmaceutical compositions for thetreatment of angiogenesis-related disorders.

In certain exemplary embodiments, there is provided a method foridentifying Migrastatin analogs having cell migration inhibitoryactivity, the method comprising steps of:

-   -   a. providing a plurality of cells;    -   b. applying a scratch to the cell layer surface;    -   c. contacting the cells with a compound having the structure (I)        as defined generally above and in classes and subclasses herein;        or pharmaceutically acceptable derivative thereof; and    -   b. evaluating the wound healing effect of the compound on the        cells.

In certain embodiments, the compound being contacted with the pluralityof cells is at a concentration ≦200 μM. In certain exemplaryembodiments, the compound has the following stereochemistry:

In certain embodiments, in the method described directly above, the stepof evaluating comprises comparing the compound wound healing effect withthat observed for cells exposed to a reference Migrastatinconcentration. In certain exemplary embodiments, the referenceMigrastatin concentration is about 100 μM. In certain exemplaryembodiments, the reference Migrastatin concentration is about 75 μM. Incertain exemplary embodiments, the reference Migrastatin concentrationis about 50 μM. In certain exemplary embodiments, the referenceMigrastatin concentration is about 30 μM.

In certain embodiments, the method is for identifying Migrastatinanalogs useful in the preparation of pharmaceutical compositions for thetreatment of metastasis-related disorders

In certain embodiments, the method may be adapted to high-throughputformat wherein the cells and test compounds are introduced and assayedin each of a plurality of reaction vessels. For example, in certainembodiments, there is provided a highthroughput method for identifyingMigrastatin analogs having cell migration inhibitory activity, themethod comprising steps of:

-   -   a. introducing a plurality of cells in each of a plurality of        reaction vessels;    -   b. in each reaction vessel, applying a scratch to the cell layer        surface;    -   c. contacting the cells, in each reaction vessel, with one or        more test compounds having the structure (I) as defined        generally above and in classes and subclasses herein; or        pharmaceutically acceptable derivative thereof; and    -   d. evaluating the wound healing effect of the test compound on        the cells in each reaction vessel.

In certain embodiments, the test compound being contacted with theplurality of cells is at a concentration ≦200 μM. In certain exemplaryembodiments, the test compound has the following stereochemistry:

In certain embodiments, in the method described directly above, the stepof evaluating comprises comparing the compound wound healing effect ineach reaction vessel with that observed for cells exposed to a referenceMigrastatin concentration. In certain exemplary embodiments, thereference Migrastatin concentration is about 100 μM. In certainexemplary embodiments, the reference Migrastatin concentration is about75 μM. In certain exemplary embodiments, the reference Migrastatinconcentration is about 50 μM. In certain exemplary embodiments, thereference Migrastatin concentration is about 30 μM.

In certain embodiments, the method is for identifying Migrastatinanalogs useful in the preparation of pharmaceutical compositions for thetreatment of angiogenesis-related disorders.

In certain other exemplary embodiments, there is provided a method foridentifying Migrastatin analogs having cell migration inhibitoryactivity, comprising steps of:

a. introducing a plurality of cells into an upper compartment;

b. introducing a test compound having the structure (I) as definedgenerally above and in classes and subclasses herein; orpharmaceutically acceptable derivative thereof; into the uppercompartment and a lower compartment, whereby the lower compartment isseparated from the upper compartment by a cell-permeable membrane; and

c. assessing cell migration from the upper to the lower compartmentafter a given period of time.

In certain embodiments, the compound being contacted with the pluralityof cells is at a concentration ≦200 μM. In certain exemplaryembodiments, the compound has the following stereochemistry:

In certain embodiments, in the method described directly above, the stepof evaluating comprises comparing cell migration from the upper to thelower compartment with that observed for cells exposed to a referenceMigrastatin concentration after about the same period of time. Incertain exemplary embodiments, the reference Migrastatin concentrationis about 100 μM. In certain exemplary embodiments, the referenceMigrastatin concentration is about 75 μM. In certain exemplaryembodiments, the reference Migrastatin concentration is about 50 μM. Incertain exemplary embodiments, the reference Migrastatin concentrationis about 30 μM.

In certain embodiments, the method is for identifying Migrastatinanalogs useful in the preparation of pharmaceutical compositions for thetreatment of metastasis-related disorders.

In certain embodiments, the method may be adapted to high-throughputformat wherein the cells and test compounds are introduced and assayedin each of a plurality of reaction vessels. For example, in certainembodiments, there is provided a highthroughput method for identifyingMigrastatin analogs having cell migration inhibitory activity, themethod comprising steps of:

a. providing a plurality of reaction vessels, each comprising an upperand lower compartment separated by a cell-permeable membrane;

b. introducing a plurality of cells into the upper compartment of eachof the plurality of reaction vessels;

c. introducing a test compound having the structure (I) as definedgenerally above and in classes and subclasses herein; orpharmaceutically acceptable derivative thereof; into the upper and lowercompartment of each of the plurality of reaction vessels; and

d. in each reaction vessel, assessing cell migration from the upper tothe lower compartment after a given period of time.

In certain embodiments of each of the highthroughput methods describedabove, a different concentration of the same test compound is introducedin each reaction vessel. In certain other embodiments, a different testcompound is introduced in each reaction vessel. In certain embodiments,a different concentration of the same test compound is introduced in asubset of the reaction vessels; and a different test compound isintroduced in another subset of the reaction vessels.

In certain embodiments, a highthroughput method according to the presentinvention is practiced with dense arrays of reaction vessels.Preferably, the center-to-center distance between reaction vessels isless than about 8.5 mm. More preferably, the distance is less than 4.5mm. Even more preferably the distance is less than approximately 2.25mm. Most preferably, the distance is less than approximately 1 mm. Incertain embodiments, the method is performed with a 48-well culturedish.

Conventional high throughput screens are often performed in commerciallyavailable 48- or 96-well plates (see, for example, Rice et al. Anal.Biochem. 241:254-259. 1996). Such plates may be utilized according tothe present invention. However, denser arrays are generally preferred,though it is appreciated that such arrays may desirably have the sameexternal dimensions of a standard 48- or 96-well plate in order tofacilitate automation using available equipment. Plates containing 384(Nalge Nunc International, Naperville, Ill.; Greiner America, Lake Mary,Fla.; Corning Costar, Corning, N.Y.) or 1536 (Greiner America, LakeMary, Fla.) wells have recently become commercially available and may beused in the practice of the present invention. In certain embodiments, ahighthroughput method according to the present invention is compatiblewith any or all of these array formats.

Pharmaceutical Uses and Methods of Treatment

In yet another aspect, the present invention provides methods oftreatment of various disorders, including those associated withmetastasis and/or increased angiogenic activity. In certain embodiments,according to the methods of treatment of the present invention,metastasis and/or the growth of tumor cells is inhibited by contactingsaid tumor cells with an inventive compound or composition, as describedherein.

Accordingly, in another aspect of the invention, methods for thetreatment of cancer are provided comprising administering atherapeutically effective amount of a compound of formula (I), asdescribed herein, to a subject in need thereof. In certain embodiments,a method for the treatment of cancer is provided comprisingadministering a therapeutically effective amount of an inventivecompound, or a pharmaceutical composition comprising an inventivecompound to a subject in need thereof, in such amounts and for such timeas is necessary to achieve the desired result.

In certain embodiments, the method involves the administration of atherapeutically effective amount of the compound or a pharmaceuticallyacceptable derivative thereof to a subject (including, but not limitedto a human or animal) in need of it. In certain embodiments, theinventive compounds as useful for the treatment of cancer (including,but not limited to, glioblastoma, retinoblastoma, breast cancer,cervical cancer, colon and rectal cancer, leukemia, lymphoma, lungcancer (including, but not limited to small cell lung cancer), melanomaand/or skin cancer, multiple myeloma, non-Hodgkin's lymphoma, ovariancancer, pancreatic cancer, prostate cancer and gastric cancer, bladdercancer, uterine cancer, kidney cancer, testicular cancer, stomachcancer, brain cancer, liver cancer, or esophageal cancer).

As discussed above, the compounds of the present invention are inhibitmetastasis of tumor cells and/or inhibiting the growth of tumor cells.In general, the inventive anticancer agents are useful in the treatmentof cancers and other proliferative disorders, including, but not limitedto breast cancer, cervical cancer, colon and rectal cancer, leukemia,lung cancer, melanoma, multiple myeloma, non-Hodgkin's lymphoma, ovariancancer, pancreatic cancer, prostate cancer, and gastric cancer, to namea few. In certain embodiments, the inventive anticancer agents areactive against leukemia cells and melanoma cells, and thus are usefulfor the treatment of leukemias (e.g., myeloid, lymphocytic, myelocyticand lymphoblastic leukemias) and malignant melanomas. In still otherembodiments, the inventive anticancer agents are active against solidtumors.

In certain embodiments, the present invention provides a method forpreventing metastasis of tumor cells in a subject comprisingadministering to a subject (including, but not limited to, a human oranimal) in need thereof a therapeutically effective amount of a compoundof the invention and a pharmaceutically acceptable carrier, adjuvant orvehicle. In certain exemplary embodiments, the method is used to preventmetastasis of prostate, breast, colon, bladder, cervical, skin,testicular, kidney, ovarian, stomach, brain, liver, pancreatic oresophageal cancer or lymphoma, leukemia, or multiple myeloma, to name afew.

In another aspect, the present invention provides methods for decreasingmigration of tumor cells. In a further aspect, the present inventionprovides methods for decreasing anchorage-independent growth of tumorcells. In yet a further aspect, the present invention provides methodsfor inhibiting angiogenesis.

In yet another aspect, the present invention provides methods forpreventing unwanted angiogenesis in a subject (including, but notlimited to, a human or animal).

As used herein, the term “angiogenesis” means the generation of newblood vessels into a tissue or organ. Under normal physiologicalconditions, humans or animals only undergo angiogenesis in very specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fetal and embryonal development and formation of thecorpus luteum, endometrium and placenta. The control of angiogenesis isa highly regulated system of angiogenic stimulators and inhibitors. Thecontrol of angiogenesis has been found to be altered in certain diseasestates and, in many cases, the pathological damage associated with thedisease is related to the uncontrolled angiogenesis.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel. In the disease state,prevention of angiogenesis could avert the damage caused by the invasionof the new microvascular system.

Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, tumor metastasis and abnormal growth by endothelial cells andsupports the pathological damage seen in these conditions. The diversepathological states created due to unregulated angiogenesis have beengrouped together as angiogenic dependent or angiogenic associateddiseases. Therapies directed at control of the angiogenic processescould lead to the abrogation or mitigation of these diseases.

One example of a disease involving an angiogenic process is ocularneovascular disease. This disease is characterized by invasion of newblood vessels into the structures of the eye such as the retina orcornea. It is the most common cause of blindness and is involved inapproximately twenty eye diseases. In age-related macular degeneration,the associated visual problems are caused by an ingrowth of chorioidalcapillaries through defects in Bruch's membrane with proliferation offibrovascular tissue beneath the retinal pigment epithelium. Angiogenicdamage is also associated with diabetic retinopathy, retinopathy ofprematurity, corneal graft rejection, neovascular glaucoma andretrolental fibroplasia. Other diseases associated with cornealneovascularization include, but are not limited to, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginaldegeneration, mariginal keratolysis, rheumatoid arthritis, systemiclupus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis,Stevens-Johnson disease, pemphigoid, radial keratotomy, and cornealgraph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget'sdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, Eales'disease, Behcet's disease, infections causing a retinitis orchoroiditis, presumed ocular histoplasmosis, Best's disease, myopia,optic pits, Stargardt's disease, pars planitis, chronic retinaldetachment, hyperviscosity syndromes, toxoplasmosis, trauma andpost-laser complications. Other diseases include, but are not limitedto, diseases associated with rubeosis (neovasculariation of the angle)and diseases caused by the abnormal proliferation of fibrovascular orfibrous tissue including all forms of proliferative vitreoretinopathy.

Another disease in which angiogenesis is believed to be involved isrheumatoid arthritis. The blood vessels in the synovial lining of thejoints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.

Factors associated with angiogenesis may also have a role inosteoarthritis. The activation of the chondrocytes by angiogenic-relatedfactors contributes to the destruction of the joint. At a later stage,the angiogenic factors would promote new bone formation. Therapeuticintervention that prevents the bone destruction could halt the progressof the disease and provide relief for persons suffering with arthritis.

Chronic inflammation may also involve pathological angiogenesis. Suchdisease states as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells. Another pathological roleassociated with angiogenesis is found in atherosclerosis. The plaquesformed within the lumen of blood vessels have been shown to haveangiogenic stimulatory activity.

One of the most frequent angiogenic diseases of childhood is thehemangioma. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, the hemangiomatoses, have a highmortality rate. Therapy-resistant hemangiomas exist that cannot betreated with therapeutics currently in use.

Angiogenesis is also responsible for damage found in hereditary diseasessuch as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding and sometimes with pulmonaryor hepatic arteriovenous fistula.

Angiogenesis is prominent in solid tumor formation and metastasis.Angiogenic factors have been found associated with several solid tumorssuch as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma,and osteosarcoma. A tumor cannot expand without a blood supply toprovide nutrients and remove cellular wastes. Tumors in whichangiogenesis is important include solid tumors, and benign tumors suchas acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.Prevention of angiogenesis could halt the growth of these tumors and theresultant damage to the animal due to the presence of the tumor.

It should be noted that angiogenesis has been associated with blood-borntumors such as leukemias, any of various acute or chronic neoplasticdiseases of the bone marrow in which unrestrained proliferation of whiteblood cells occurs, usually accompanied by anemia, impaired bloodclotting, and enlargement of the lymph nodes, liver, and spleen. It isbelieved that angiogenesis plays a role in the abnormalities in the bonemarrow that give rise to leukemia-like tumors.

Angiogenesis is important in two stages of tumor metastasis. The firststage where angiogenesis stimulation is important is in thevascularization of the tumor which allows tumor cells to enter the bloodstream and to circulate throughout the body. After the tumor cells haveleft the primary site, and have settled into the secondary, metastasissite, angiogenesis must occur before the new tumor can grow and expand.Therefore, prevention of angiogenesis could lead to the prevention ofmetastasis of tumors and possibly contain the neoplastic growth at theprimary site.

Knowledge of the role of angiogenesis in the maintenance and metastasisof tumors has led to a prognostic indicator for breast cancer. Theamount of neovascularization found in the primary tumor was determinedby counting the microvessel density in the area of the most intenseneovascularization in invasive breast carcinoma. A high level ofmicrovessel density was found to correlate with tumor recurrence.Control of angiogenesis by therapeutic means could possibly lead tocessation of the recurrence of the tumors.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimentalside effect of surgical procedures and may be caused or exacerbated byangiogenesis. Adhesions are a frequent complication of surgery and leadto problems such as small bowel obstruction.

Accordingly, in one aspect, the present invention provides method toinhibit unwanted angiogenesis in a subject (including, but not limitedto, a human or animal).

In another aspect, the present invention provides a method for thetreatment for diseases mediated by angiogenesis.

In another aspect, the present invention provides a method for thetreatment for macular degeneration.

In another aspect, the present invention provides a method for thetreatment for all forms of proliferative vitreoretinopathy includingthose forms not associated with diabetes.

In another aspect, the present invention provides a method for thetreatment for solid tumors.

In another aspect, the present invention provides a method for thetreatment of blood-borne tumors, such as leukemia.

In another aspect, the present invention provides a method for thetreatment of hemangioma.

In another aspect, the present invention provides a method for thetreatment of retrolental fibroplasia.

In another aspect, the present invention provides a method for thetreatment of psoriasis.

In another aspect, the present invention provides a method for thetreatment of Kaposi's sarcoma.

In another aspect, the present invention provides a method for thetreatment of Crohn's disease.

In another aspect, the present invention provides a method for thetreatment of diabetic retinopathy.

Thus, in certain embodiments, the invention provides a method forpreventing unwanted angiogenesis in a subject (including, but notlimited to, a human or animal) comprising administering to a subject inneed thereof a therapeutically effective amount of the compound of theinvention in an amount effective to inhibit angiogenesis.

In certain other embodiments, the invention provides a method fortreating an angiogenesis-dependent disease in a subject (including, butnot limited to, a human or animal) comprising administering to a subjectin need thereof a therapeutically effective amount of the compound ofthe invention in an amount effective to inhibit angiogenesis.

Diseases associated with corneal neovascularization that can be treatedaccording to the present invention include but are not limited to,diabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma and retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical bums, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginaldegeneration, mariginal keratolysis, trauma, rheumatoid arthritis,systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis,Stevens-Johnson disease, pemphigoid, radial keratotomy, and cornealgraph rejection.

Diseases associated with retinal/choroidal neovascularization that canbe treated according to the present invention include, but are notlimited to, diabetic retinopathy, macular degeneration, sickle cellanemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales' disease,Behcet's disease, infections causing a retinitis or choroiditis,presumed ocular histoplasmosis, Best's disease, myopia, optic pits,Stargardt's disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma and post-lasercomplications. Other diseases include, but are not limited to, diseasesassociated with rubeosis (neovasculariation of the angle) and diseasescaused by the abnormal proliferation of fibrovascular or fibrous tissueincluding all forms of proliferative vitreoretinopathy, whether or notassociated with diabetes.

Diseases associated with chronic inflammation can be treated by thecompositions and methods of the present invention. Diseases withsymptoms of chronic inflammation include inflammatory bowel diseasessuch as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosisand rheumatoid arthritis. Angiogenesis is a key element that thesechronic inflammatory diseases have in common. The chronic inflammationdepends on continuous formation of capillary sprouts to maintain aninflux of inflammatory cells. The influx and presence of theinflammatory cells produce granulomas and thus, maintains the chronicinflammatory state Inhibition of angiogenesis by the compositions andmethods of the present invention would prevent the formation of thegranulomas and alleviate the disease.

The compositions and methods of the present invention can be used totreat patients with inflammatory bowel diseases such as Crohn's diseaseand ulcerative colitis. Both Crohn's disease and ulcerative colitis arecharacterized by chronic inflammation and angiogenesis at various sitesin the gastrointestinal tract. Crohn's disease is characterized bychronic granulomatous inflammation throughout the gastrointestinal tractconsisting of new capillary sprouts surrounded by a cylinder ofinflammatory cells. Prevention of angiogenesis by the compositions andmethods of the present invention inhibits the formation of the sproutsand prevents the formation of granulomas.

Crohn's disease occurs as a chronic transmural inflammatory disease thatmost commonly affects the distal ileum and colon but may also occur inany part of the gastrointestinal tract from the mouth to the anus andperianal area. Patients with Crohn's disease generally have chronicdiarrhea associated with abdominal pain, fever, anorexia, weight lossand abdominal swelling. Ulcerative colitis is also a chronic,nonspecific, inflammatory and ulcerative disease arising in the colonicmucosa and is characterized by the presence of bloody diarrhea.

The inflammatory bowel diseases also show extraintestinal manifestationssuch as skin lesions. Such lesions are characterized by inflammation andangiogenesis and can occur at many sites other than the gastrointestinaltract. The compositions and methods of the present invention are alsocapable of treating these lesions by preventing the angiogenesis, thusreducing the influx of inflammatory cells and the lesion formation.

Sarcoidosis is another chronic inflammatory disease that ischaracterized as a multisystem granulomatous disorder. The granulomas ofthis disease may form anywhere in the body and thus the symptoms dependon the site of the granulomas and whether the disease active. Thegranulomas are created by the angiogenic capillary sprouts providing aconstant supply of inflammatory cells.

The compositions and methods of the present invention can also treat thechronic inflammatory conditions associated with psoriasis. Psoriasis, askin disease, is another chronic and recurrent disease that ischaracterized by papules and plaques of various sizes. Prevention of theformation of the new blood vessels necessary to maintain thecharacteristic lesions leads to relief from the symptoms.

Another disease which can be treated according to the present inventionis rheumatoid arthritis. Rheumatoid arthritis is a chronic inflammatorydisease characterized by nonspecific inflammation of the peripheraljoints. It is believed that the blood vessels in the synovial lining ofthe joints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.Another disease that can be treated according to the present inventionare hemangiomas, Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia, solid or blood borne tumors and acquired immunedeficiency syndrome.

It will be appreciated that the compounds and compositions, according tothe method of the present invention, may be administered using anyamount and any route of administration effective for the treatment ofcancer and/or disorders associated with metastasis and/or angiogenesis.Thus, the expression “effective amount” as used herein, refers to asufficient amount of agent to inhibit the growth of tumor cells, orrefers to a sufficient amount to reduce the effects of cancer. The exactamount required will vary from subject to subject, depending on thespecies, age, and general condition of the subject, the severity of thediseases, the particular anticancer agent, its mode of administration,and the like.

The compounds of the invention are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof therapeutic agent appropriate for the patient to be treated. It willbe understood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient ororganism will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see, for example, Goodmanand Gilman's, “The Pharmacological Basis of Therapeutics”, TenthEdition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press,155-173, 2001, which is incorporated herein by reference in itsentirety).

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier, adjuvant or vehicle in a desired dosage, thepharmaceutical compositions of this invention can be administered tohumans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, creams or drops), bucally, as an oral or nasalspray, or the like, depending on the severity of the infection beingtreated. In certain embodiments, compounds of the invention may beadministered at dosage levels of about 0.001 mg/kg to about 50 mg/kg,from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about50 mg/kg, from about 1 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 1 mg/kg to about 40 mg/kg, from about 0.1mg/kg to about 30 mg/kg, from about 1 mg/kg to about 30 mg/kg, fromabout 5 mg/kg to about 30 mg/kg, from about 0.1 mg/kg to about 20 mg/kg,from about 1 mg/kg to about 20 mg/kg, of subject body weight per day,one or more times a day, to obtain the desired therapeutic effect. Itwill also be appreciated that dosages smaller than 0.001 mg/kg orgreater than 50 mg/kg (for example 50-100 mg/kg) can be administered toa subject. In certain embodiments, compounds are administered orally orparenterally.

Treatment Kit

In other embodiments, the present invention relates to a kit forconveniently and effectively carrying out the methods in accordance withthe present invention. In general, the pharmaceutical pack or kitcomprises one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention. Suchkits are especially suited for the delivery of solid oral forms such astablets or capsules. Such a kit preferably includes a number of unitdosages, and may also include a card having the dosages oriented in theorder of their intended use. If desired, a memory aid can be provided,for example in the form of numbers, letters, or other markings or with acalendar insert, designating the days in the treatment schedule in whichthe dosages can be administered. Alternatively, placebo dosages, orcalcium dietary supplements, either in a form similar to or distinctfrom the dosages of the pharmaceutical compositions, can be included toprovide a kit in which a dosage is taken every day. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Equivalents

The representative examples that follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. It should further be appreciated that the contents ofthose cited references are incorporated herein by reference to helpillustrate the state of the art. Throughput this document, variouspublications are referred to, each of which is hereby incorporated byreference in its entirety in an effort to more fully describe the stateof the art to which the invention pertains.

The following examples contain important additional information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and the equivalents thereof.

EXEMPLIFICATION

The compounds of this invention and their preparation can be understoodfurther by the examples that illustrate some of the processes by whichthese compounds are prepared or used. It will be appreciated, however,that these examples do not limit the invention. Variations of theinvention, now known or further developed, are considered to fall withinthe scope of the present invention as described herein and ashereinafter claimed.

1) General Description of Synthetic Methods:

The practitioner has a a well-established literature of macrolidechemistry to draw upon, in combination with the information containedherein, for guidance on synthetic strategies, protecting groups, andother materials and methods useful for the synthesis of the compounds ofthis invention.

The various references cited herein provide helpful backgroundinformation on preparing compounds similar to the inventive compoundsdescribed herein or relevant intermediates, as well as information onformulation, uses, and administration of such compounds which may be ofinterest.

Moreover, the practitioner is directed to the specific guidance andexamples provided in this document relating to various exemplarycompounds and intermediates thereof.

The compounds of this invention and their preparation can be understoodfurther by the examples that illustrate some of the processes by whichthese compounds are prepared or used. It will be appreciated, however,that these examples do not limit the invention. Variations of theinvention, now known or further developed, are considered to fall withinthe scope of the present invention as described herein and ashereinafter claimed.

According to the present invention, any available techniques can be usedto make or prepare the inventive compounds or compositions includingthem. For example, a variety of solution phase synthetic methods such asthose discussed in detail below may be used. Alternatively oradditionally, the inventive compounds may be prepared using any of avariety combinatorial techniques, parallel synthesis and/or solid phasesynthetic methods known in the art.

It will be appreciated as described below, that a variety of inventivecompounds can be synthesized according to the methods described herein.The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCompany (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis,Mo.), or are prepared by methods well known to a person of ordinaryskill in the art following procedures described in such references asFieser and Fieser 1991, “Reagents for Organic Synthesis”, vols 1-17,John Wiley and Sons, New York, N.Y., 1991; Rodd 1989 “Chemistry ofCarbon Compounds”, vols. 1-5 and supps, Elsevier Science Publishers,1989; “Organic Reactions”, vols 1-40, John Wiley and Sons, New York,N.Y., 1991; March 2001, “Advanced Organic Chemistry”, 5th ed. John Wileyand Sons, New York, N.Y.; and Larock 1990, “Comprehensive OrganicTransformations: A Guide to Functional Group Preparations”, 2^(nd) ed.VCH Publishers. These schemes are merely illustrative of some methods bywhich the compounds of this invention can be synthesized, and variousmodifications to these schemes can be made and will be suggested to aperson of ordinary skill in the art having regard to this disclosure.

The starting materials, intermediates, and compounds of this inventionmay be isolated and purified using conventional techniques, includingfiltration, distillation, crystallization, chromatography, and the like.They may be characterized using conventional methods, including physicalconstants and spectral data.

Certain exemplary compounds of the invention are listed below:

As discussed herein, cancer chemotherapy traditionally relies ontherapeutic agents with cytotoxic properties that inhibit tumor cellproliferation and cause cell death. Recently, the idea of targeting cellmigration as an alternative strategy for the development of anti-cancertherapies has generated considerable interest.¹ Intense research effortsare currently directed to the exploration of cell shape change andmovement and their underlying mechanisms.² Cell migration is involved ina number of physiological processes, including ovulation, embryonicdevelopment, tissue regeneration (wound healing), and inflammation. Onthe other hand, cell migration is also observed in pathologicalconditions such as tumor angiogenesis, cancer cell invasion, andmetastasis.³ It is believed that primary solid tumors depend onangiogenesis (formation of new blood vessels) to obtain the necessaryoxygen and nutrient supplies for growth beyond a certain size (ca. 1-2mm) The transition from a pre-angiogenic condition to tumorangiogenesis,⁴ often referred to as the angiogenic switch, is followedby tumor growth, cancer cell invasion, and metastasis.⁵ In principle, itcould be possible to halt (or retard) this procession at differentstages with the help of cell migration inhibitors. Since cell migrationunder ordinary physiological conditions in adults is rather infrequent,its repression might be accompanied by manageable toxicity.

A significant part of our general research program focuses on thedevelopment of novel, natural product-inspired anti-cancer agents. Theseefforts have led to the total chemical synthesis of a number ofprominent anti-tumor natural products, such as the epothilones,⁶Taxol®,⁷ and most recently, radicicol⁸ and TMC-95A/B.⁹ The recent entryof 12,13-desoxyepothilone B (dEpoB), first prepared by total chemicalsynthesis, into phase II clinical trials:⁶ has been followed by thediscovery of a new generation of highly potent epothilone analogs.¹¹ Forthe most part, our endeavors have converged on cytotoxic agents. Thepossibility of exploiting natural products as leads for the developmentof anti-angiogenic and anti-metastatic agents was prompted by the recentisolation and synthesis of compounds such as epoxyquinol A and B,¹²trachyspic acid,¹³ azaspirene,¹⁴ evodiamine,¹⁵ motuporamines,¹⁶borrelidin,¹⁷ and terpestacin.¹⁸

In particular, a series of independent reports by Imoto¹⁹ and KosanBioscience researchers²⁰ on the discovery of the natural productmigrastatin (1) enhanced our interest in this area (Scheme 16). It wasreported that 1, isolated from a cultured broth of Streptomyces, has thepotential of metastasis suppression through its ability to inhibit tumorcell migration. Although the reported activity of migrastatin in a woundhealing assay was rather modest (IC₅₀ value of 29 μM), we considered itas an attractive lead compound in the search for other, more potentagents. The structure of migrastatin (1), determined by X-ray crystalstructure analysis, features a 14-membered macrolactone with acharacteristic glutarimide-containing side chain. Embedded in themacrocycle are a trisubstituted (Z)-alkene and two disubstituted(E)-alkenes, as well as three contiguous stereocenters. The side chainprojecting from the cyclic core is associated with stereogenic centersat C13 and C14.

Upon reviewing the literature in search of glutarimide-containingnatural products, prominent examples such as cycloheximide (CHX),²¹streptimidone,²² and thalidomide (which has resurfaced recently as ananti-angiogenic agent despite its controversial history²³) can beidentified. Moreover, a number of structural homologs of migrastatin,namely lactimidomycin,²⁴ dorrigocin A and B,^(20,25) isomigrastatin,²⁰and NK30424A/B,²⁶ have been discovered (Scheme 16). In 1992,lactimidomycin was isolated from Streptomyces amphibiosporus andcharacterized by researchers at Bristol-Myers Squibb. This uniquetriene-containing 12-membered macrolactone antibiotic is highlycytotoxic in vitro against a number of tumor cell lines and displays invivo anti-tumor activity in mice. In addition, lactimidomycin exhibitspotent anti-fungal properties and acts as an inhibitor of DNA andprotein synthesis. Two years later, the isolation of dorrigocin A andits allylic isomer dorrigocin B from Streptomyces platensis wasdescribed by researchers at Abbott Laboratories. The dorrigocins arelinear polyketide carboxylic acids with a functional group arrangementclosely related to migrastatin and isomigrastatin (see below),respectively. They were found to reverse the morphology ofras-transformed NIH/3T3 cells from a transformed phenotype to a normalone. Dorrigocin A was also reported to be the first natural productinhibitor of the carboxyl methyltransferase involved in Ras processing.In 2002, the dorrigocins were again isolated from Streptomyces platensisby researchers at Kosan Biosciences along with migrastatin and a newmember of the family, isomigrastatin. Structurally, isomigrastatin canbe described as being derived from migrastatin via an allylictransposition (C13→C11) and a concomitant double bond isomerization.Thus, isomigrastatin is a 12-membered macrolactone with an exocyclictrisubstituted (E)-alkene. The Kosan researchers have shown that thehydrolysis of isomigrastatin leads to dorrigocin B, whereas thehydrolysis of migrastatin produces a geometric isomer of dorrigocin A.The biological profile of isomigrastatin has not been reported to date.The latest members of the glutarimide-containing macrolide family arethe natural products NK30424A and its stereoisomer NK30424B, isolatedfrom Streptomyces sp. NA30424 by researchers at Nippon Kayaku.Furthermore, four related compounds, derived from oxidation of thethioether to the sulfoxide, were detected as minor constituents in thecultured broth and were titled as NK30424AS1-2 and NK30424BS1-2. The NKcompounds are formally derived from isomigrastatin by conjugate additionof cysteine to the C2-C3 double bond and hydroxylation at C17.Interestingly, these NK congeners are reported to be very potentinhibitors of lipopolysaccharide-induced tumor necrosis factor-α (TNF-α)promoter activity. To date, migrastatin is the only member of thenatural product family described above in which the relative andabsolute configurations have been determined. Possibly, total chemicalsynthesis might aid in deciphering the stereochemistry of other membersof this series.

In one aspect, the present invention provides synthetic methods forpreparing migrastatin and analogs thereof. The first asymmetric totalsynthesis of naturally occurring (+)-migrastatin (1) is describedherein. Exemplary syntheses of Migrastatin analogs are also describedherein. The total synthesis of 1 provides researchers (includingApplicant) access to material for an independent evaluation of thebiology of the natural product and with opportunities via standardmedicinal chemistry for gaining access to a broad range of structuralanalogs. Moreover, the present invention provides synthetic methodsallowing access to a variety of Migrastatin analogs and the explorationof structural types that could not, plausibly, be accessible by chemicalmodification of migrastatin itself. From a practical standpoint, invitro screening of migrastatin derivatives (e.g., in cell migrationassays) may well lead to informative structure-activity relationship(SAR) profiles, conceivably assisting in the emergence of compounds withimproved biological profiles for progression to in vivo models.Preliminary SAR trends are provided herein. In addition, effortsdirected at target identification are expected to yield some insightinto the biological mode of action of migrastatin and its congeners andanalogs.

As discussed above, in one aspect, the present invention providesmethods for the preparation of Migrastatin and analogs thereof. Detailedbelow is a synthetic approach, which resulted in an efficient andflexible total synthesis of 1. Additional guidance may be found, forexample, in Gaul, C. et al.; J. Am. Chem. Soc. 2003, 125, 6042; Gaul, C.et al.; J. Am. Chem. Soc. 2004, 126(4), 1038-1040; each of which ishereby incorporated by reference in its entirety; and Gaul, C. et al.;J. Am. Chem. Soc. 2004, 126 (36), 11326-11337. Migrastatin having knownbiological activity, it was expected that its analogs would exhibitsimilar activity. As discussed above, however, the present inventionprovides the ability to synthesize various migrastatin analogs with avariety of structural features; thereby allowing one to probe andevaluate Structure-Activity Relationships trends within this class ofmacrocyclic compounds. Preliminary SAR studies²⁸ have been reported. Forexample, guidance may be found in U.S. Provisional Application Nos.:60/458,827 filed Mar. 28, 2003 and 60/496,165 filed Aug. 19, 2003; eachof which are incorporated herein by reference. In a preliminary study, afew migrastatin analogs were evaluated in both tube formation and woundhealing assay (See Example 52 and Tables 1 and 2). In addition to twoanalogs closely structurally related to migrastatin (i.e.,N-Methyl-migrastatin and 2,3-Dihydromigrastatin (41)), the question ofhow the migrastatin C-13 side chain might impact activity wasinvestigated (cf. Migrastatin-Core (45)). One advantage of this type ofcompounds lies in the simplicity of their structure; they are thereforeeasier to synthesize, less costly and more amenable to large scalepreparation. Compound 45, along with the other two migrastatin analogswere thus subjected to the aforementioned assays. A chamber cellmigration assay was also proposed that could be used to screen andidentify migrastatin analogs exhibiting cell migration inhibitoryactivity (See Example 52 and Table 3). Preliminary results aresummarized in Tables 1-3 below.

TABLE 1 Tube formation assay Substance Minimum effect concentrationMigrastatin (1) 100 μM N-Methyl-migrastatin 200 μM2,3-Dihydromigrastatin (41)  50 μM Migrastatin-Core (45)  10 μM Testedconcentrations: 200, 100, 50, 25, 10 μM

TABLE 2 Scratch Assay Substance Minimum effect concentration Migrastatin(1) 100 μM N-Methyl-migrastatin 100 μM 2,3-Dihydromigrastatin (41)  25μM Tested concentrations: 200, 100, 50, 25, 10 μM

TABLE 3 Chamber Assay Substance IC₅₀ Migrastatin (1) 200 μM Testedconcentrations: 200, 100, 50, 25, 10 μM

Based on the aforementioned preliminary biological data, and withoutwishing to be bound to any particular theory, we proposed that“truncated” migrastatin analogs (i.e., analogs lacking the side chain atC-13, or having a significantly shorter side chain at C-13) may exhibitimproved therapeutic activity. For example, compounds such as thosehaving the general structures depicted below were expected to show goodactivity as cell migration inhibitors and/or angiogenesis inhibitors:

wherein n and R₃-R₆ are as defined in classes and subclasses herein; andY₁ and Y₂ are independently hydrogen, an aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aryl or heteroaryl moiety; or —WR^(Y1);wherein W is independently —O—, —S— or —NR^(Y2)—, wherein eachoccurrence of R^(Y1) and R^(Y2) is independently hydrogen, or analiphatic, heteroaliphatic, alicyclic, heteroalicyclic, aryl orheteroaryl moiety; or Y₁ and Y₂ together with the carbon atom to whichthey are attached form a moiety having the structure:

In order to evaluate this hypothesis further, additional analogs weresynthesized and tested. Compounds were tested in chamber cell migration,cell proliferation and wound healing assays (see Examples 53, 55 and 56,respectively), results of which confirmed our initial hypothesis. Cellmigration inhibitory activity of compounds of the invention wasevaluated with 4T1 mouse breast tumor cells in chamber cell migrationand wound healing assays. The highly aggressive and invasive 4T1 cellsare routinely used as model for evaluating test compounds for thetreatment of human breast cancer, because the progessive spread of 4T1cells to lymph nodes, lungs and other organs can be seen to mimicmetastasis of human mammary cancer.

In the wound healing assay, standard scratches (i.e., wounds) were madethrough a confluent 4T1 cell layer. In the absence of serum, cells wouldnot migrate across the empty space created by application of thescratch. Upon addition of serum containing migration-enabling factors,migration of 4T1 cells across the scratch was induced. Exposure to thesecells to inventive compounds allowed the evaluation of the cellmigration inhibitory activity of these compounds. For example, as seenin FIG. 6C, 2,3-dihydro migrastatin (48) almost completely inhibitedcell migration across the scratch, while the effect of migrastatin (1)at the same concentration (200 μM) was not as great (See, FIG. 6D).

Compounds were also subjected to mouse stability studies. As expected,macrolactone-type compounds showed lesser stability in mouse plasma thantheir macrolactam or macroketone counterparts (See, for example,stability data obtained for macrolactone compounds 45 and 48, versusthat obtained for macrolactam 55 and macroketone 60 (i.e., the lactonefunctionality is more vulnerable to esterase hydrolysis).

Finally, compounds showing very good in vitro activity (e.g., chamber4T1 and HUVEC cell migration assay) were tested in vivo in a mousebreast cancer model (See Example 57 and FIG. 4). Macroketone 60administered at 10 mg/kg exhibited ˜94% inhibition of lung metastasis.Administration of 20 mg/kg of macroketone 60 resulted in ˜99% inhibitionof lung metastasis. Similarly, Administration of 10 mg/kg and 20 mg/kgof macrolactam 55 resulted in ˜91% inhibition of lung metastasis. The invivo data confirmed the in vitro findings, thereby validating the invitro assays described herein as good predictors of therapeutic activityin vivo.

The present invention also provides a preparation and biologicalevaluation of an extended, diverse set of migrastatin analogs which ledto the discovery of highly potent cell migration inhibitors.

Exemplary Synthetic Approach. It was particularly desirable to devise aconcise, flexible, and readily scaleable synthesis, since it wouldremain for synthesis to fuel an aggressive SAR elucidation program andto provide significant quantities of materials for in vivo studies. Thisis in keeping with the notion that total synthesis was viewed as a firstmilestone of the project, rather than as an end-point. In doing so,several structural features were considered and evaluated. For example,the (E)-configuration of the C2-C3 and C6-C7 double bonds, and the(Z)-configuration of the C11-C12 double bond of the trienic lactone areimportant features of the migrastatin core. Moreover, maintaining tightstereocontrol over the dispositions of the stereogenic centers at C8,C9, C10, and C13 was significant. In addition, the introduction of theside-chain projecting from C13 and the inclusion of the stereocenter atC14, which is not part of the ring structure, were equally importantaspects of a successful synthesis of the migrastatin target, as were theincorporation of the C15 keto group as well as the 6-substitutedglutarimide at C18.

In one embodiment, a synthetic approach that embraces these structuralissues is presented in Scheme 17. As depicted in Scheme 17, aretrosynthetic analysis is based on components 2, 3, 4, 5, and 6. The(E)-geometric character of the C2-C3 double bond could be secured viarecourse to the known compound 6. An important feature of this synthesiswould be the use of aldehyde 2, bearing the methoxy-substitutedstereogenic center, ultimately to be emplaced at C8. Another importantbuilding block would be diene 3. This type of synergistically activated,dibranched, bisoxygenated butadiene was part of our all-carbonDiels-Alder research in the mid 1970's.²⁹ Indeed, in the 1980's thistype of diene was used in the context of our LACDAC chemistry to createdihydropyrans.³⁰ The aldehyde in this case would be the previouslydiscussed 2. Appropriate disconnection of the pyran would expose thefour carbon segment comprising C10 through C13. The two methyl-branchingelements of 3 would appear at C10 and C12 in migrastatin followingappropriate manipulations. At the outset, the precise nature of the Rfunction in keto building block 4 awaited specification. A decisivecriterion for various candidate structures that might be contemplatedwould be their amenability to linkage to the emerging C13 in the contextof macrolactone formation, while enabling smooth incorporation of theδ-branched glutarimide. We note that the sum of fragments 2 and 6contains two carbons in excess of those required for formation of the14-membered macrolactone. Such a disconnection invited the prospect ofestablishing this lactone through a ring-closing metathesis (RCM)reaction with extrusion of the two seemingly extraneous carbon centers.A more detailed analysis of synthetic issues appears in the context ofthe next section, in which we describe the implementation of the broadplan.

Model Study. It seemed prudent to assess, in the context of a modelstudy, the feasibility of RCM to construct the 14-membered ring ofmigrastatin (Scheme 18).³¹ In this connection, we would also address thestereoselectivity (geometry of the C6-C7 double bond) andchemoselectivity (undesired RCM-participation of the C2-C3 and C11-C12double bonds) of the ring-closing reaction. Such questions were to befirst posed in a study directed to the synthesis of the migrastatin corestructure lacking the glutarimide-containing side chain. Since we wereconcerned about the stability, stereochemical integrity, and potentialvolatility of the previously unknown α-methoxy-α-vinyl aldehyde 2(Scheme 17) contemplated for the LACDAC reaction, we began, in thistesting phase, with the structurally less challenging heterodienophilicsiloxy-aldehyde 8 (Scheme 18). This compound was prepared fromcommercially available (S)-3-benzyloxy-1,2-propanediol 7³² in foursecurely precedented steps. The sterically demanding TBDPS protectinggroup was deliberately chosen with a view toward suppressing a possibleβ-chelation pathway relative to the desired α-chelation mode in theLACDAC sequence. Earlier research from Reetz³³ provided a suggestionthat oxygen chelation effects in the control of diastereofacialreactions are suppressed in bulky silylether settings.

Indeed, as intended, reaction of aldehyde 8 and diene 3³⁴ under theinfluence of TiCl₄ yielded the α-chelation controlled product 9 (Scheme18).³⁵ Treatment of dihydropyrone 9 with NaBH₄ and CeCl₃.7H₂O (Luchereduction)³⁶ led to the corresponding 1,2-reduced compound, whichunderwent a Ferrier rearrangement³⁷ in aqueous acidic THF to producelactol 10, with the desired (Z)-olefin now in place. Reductive openingof lactol 10 with LiBH₄ afforded diol 11 in 55% overall yield fromdihydropyrone 9. The primary hydroxyl group of 11 was selectivelyacylated with 2,6-heptadienoyl chloride 12³⁸ and, thereafter, thesecondary hydroxyl group in the acylation product was protected as itsMOM ether.

The RCM precursor 14 was reached from 13 through a three step sequence,consisting of deprotection, oxidation, and Tebbe olefination.³⁹ Whentetraene 14 was subjected to the action of Grubbs catalyst 16⁴⁰ (seestructures below) in refluxing toluene,⁴¹ the 14-membered macrolactone15 was generated as the desired (E)-congener in 50% yield. Competitiveparticipation of the electron-poor C2-C3 double bond and the stericallyhindered C11-C12 double bond in the metathesis step could not bedetected. Interestingly, treatment of 14 with Grubbs-I catalyst 17 inrefluxing CH₂Cl₂ led exclusively to the dimeric product derived fromcross metathesis of the terminal double bond of the acyl moiety.

Synthesis of Intermediate 26. The model study demonstrated the efficacyof the LACDAC sequence to construct the three stereocenters C8-C10 andthe power of RCM to establish the macrocyclic system. Encouraged bythese early results, we embarked on the total synthesis of migrastatinitself. Prior to facing the unresolved issues of building up theremaining stereocenters at C13 and C14 in the context of emplacement ofthe glutarimide moiety, we addressed an issue of process, asking thequestion whether α-methoxy-α-vinyl aldehyde 2 might indeed serve as asuitable heterodienophile in the reaction with diene 3 after all.Utilization of aldehyde 2 in the LACDAC reaction would allow us tostreamline the synthesis in a most useful way by avoiding the chemistryneeded to incorporate the C6-C7 double bond required for RCM.

Happily, we could gain an excellent entry into this type of aldehyde,starting from commercially available dimethyl2,3-O-isopropylidene-L-tartrate 18 (Scheme 19). Toward this end,tartrate 18 was reduced by DIBALH to the corresponding dialdehyde, whichwas then reacted in situ with divinylzinc to afford carbinol 19 in ahighly stereoselective fashion.⁴² Dimethylation and cleavage of theacetonide protecting group with aqueous acid furnished 1,2-diol 20 inexcellent yield.⁴³ The desired α-methoxy-α-vinyl aldehyde 2 emergedfollowing cleavage of the glycol linkage of 20. Importantly, no attemptswere undertaken to isolate 2 in neat form. Instead, a stock solution ofthe aldehyde as obtained from the glycol cleavage was directly used forthe LACDAC sequence. We were rather encouraged to find that theα-chelation-controlled cyclocondensation of 2 with butadiene 3 occurredin very good yield, producing dihydropyrone 21 as the only detecteddiastereomer. Compound 21 not only possesses the three contiguousstereocenter of the macrolide, but it also serves as a template for theconstruction of the trisubstituted C11-C12 (Z)-alkene (Scheme 20). Froma process standpoint, it is noteworthy that only two chromatographicpurifications were needed to obtain pure 21, rendering the sequenceamenable to scale-up.

Transformation of dihydropyrone 21 into open-chain diol 25 wasaccomplished as described for our model study (Scheme 18) using areduction-Ferrier rearrangement-reductive ring-opening protocol (Scheme20). Initially, we followed the Luche procedure for the reduction ofenones to effect the conversion of 21 to 22. Subsequently, we found thatthe addition of cerium salts was not needed in our case. In fact, allthe reductants screened led exclusively to 1,2-reduction. In the end,LiBH₄ turned out to be the reducing agent of choice based on itsassociated ease of handling and workup. When alcohol 22 was subjected tocatalytic amounts of camphorsulfonic acid (CSA) in refluxing aqueousTHF, the desired Ferrier-rearranged product 23 was obtained, togetherwith small amounts of dimeric acetal 24. It is appropriate to note thatthere are few examples of aqueous Ferrier rearrangements reported in theliterature,⁴⁴ whereas variants using alcohol-based nucleophiles arewidely encountered. Reductive opening of lactol 23 with LiBH₄ affordeddiol 25 in 53% overall yield (from dihydropyrone 21). In investigatingthe preparative aspects of the sequence 21→25, we realized that largeramounts of 24 (ca. 15%) were isolated when the Ferrier rearrangement wasconducted at higher concentrations (0.3M instead of 0.1M). Happily, wewere able to obtain single crystals of dimer 24. X-Ray analysis led to adecisive structural verification,⁴⁵ revealing the relative configurationof the three contiguous stereocenters and the geometry of the doublebond to be as predicted on the basis of our precedents. By extension,this X-ray analysis also confirms the structure of diol 25.

A next step in this synthesis of migrastatin involved differentiation ofthe two hydroxyl groups of 25. Previously, we had accomplished thissub-goal via a three step sequence, namely acetylation of the primaryhydroxyl group, silylation of the secondary hydroxyl group, andsubsequent removal of the acetate protecting group.²⁷ However, duringscale-up efforts, we observed the formation of considerable amounts ofdiacetylated product. This obstacle complicated purification proceduresand lowered the overall yield of the sequence. Fortunately, the problemcould easily be solved by initial disilylation, followed by a mild andselective deprotection, producing allylic alcohol 26 in 80% yield(Scheme 20).

Incorporation of the Glutarimide-Containing Side Chain. Astraightforward way to construct the two remaining stereocenters at C13and C14 could, in principle, be accomplished by an anti-selective aldolreaction between an aldehyde derived from 26 and an appropriatepropionyl fragment. Indeed, Dess-Martin oxidation⁴⁶ of 26 generatedangelic-type aldehyde 27 (Scheme 21). Fortunately, 27 proved to benotably resistant to (Z)→(E)-double bond isomerization or vinylogousepimerization, and, accordingly, could serve as a potential substrate inthe projected aldol construction. In our early studies, we exploredMasamune's anti-aldol protocol, which utilizes a boron enolate ofreadily available norephedrine derivatives.⁴⁷ This aldol reaction indeedworked smoothly with aldehyde 27. Nonetheless, we were particularlydrawn to a mild MgCl₂-catalyzed anti-aldol procedure that had recentlybeen disclosed by Evans.⁴⁸ In practice, aldehyde 27 reacted withpropionyl oxazolidinone 28 in the presence of MgCl₂, triethylamine, andTMSCl to afford, after treatment with TFA, the desired aldol adduct 29in 67% yield as a single diastereomer. Noteworthy, the robust reactionconditions, which tolerate the use of reagent-grade ethyl acetate andhigh substrate concentrations, are attractive features for scale-uppurposes. Since the next step of the projected total synthesis was theprotection of the C13 hydroxyl group as a TES ether, we tried toaccomplish the anti-aldol joining with TESCl instead of TMSCl.Unfortunately, the reaction was very slow under these conditions, andthe yields were far from satisfactory. Hence, we had to protect thesecondary hydroxyl group with TESCl in a separate step (29→30) (Scheme21).

Having successfully merged three of our five components, we focused nowon attaching glutarimide aldehyde 5 to the main fragment. AHorner-Wadsworth-Emmons (HWE) reaction between β-ketophosphonate 32 andaldehyde 5 appeared to be a plausible, attractive solution to thissynthetic problem (Scheme 21). Toward this end, we investigated thedirect addition of lithiated dimethyl methylphosphonate to imide 30 toaccess the desired phosphonate 32 in a single transformation.Unfortunately, this projected (but unprecedented) transformation metwith no success, resulting in recovery of starting material.⁴⁹Accordingly, the chiral auxiliary was removed reductively (30→31).Progress continued with a simple and reliable three stepoxidation-addition-reoxidation protocol, cleanly affording phosphonate32. Glutarimide aldehyde 5,⁵⁰ the fourth component in our syntheticplan, was then treated with phosphonate 32 using the Masamune-Roushvariant of the HWE reaction.⁵¹ Enone 33 was obtained as a single olefinisomer in excellent yield (Scheme 21). Fortunately, neither thisreaction nor any of the subsequent transformations required protectionof the glutarimide nitrogen. Conjugate reduction of enone 33 with theStryker reagent⁵² and cleavage of the TES protecting group occurredsmoothly to give alcohol 34. At this stage, the path was clear forintroduction of our last component, 2,6-heptadienoic acid 6.

Completion of a Total Synthesis of Migrastatin. In our planning stages,we presumed that acylation of secondary alcohol 34 with acid 6 would bestraightforward. Unexpectedly, a number of acylation conditions had tobe explored to accomplish the desired transformation effectively. Onlyafter several trial attempts did we find that a modified Yamaguchiacylation protocol⁵³ (using pyridine instead of DMAP) providedsatisfying yields of acylated product 35 (Scheme 22). Most otherstandard ester formation protocols (a: acid chloride+DMAP, pyridine, orAgCN, b: acid+EDC or DCC, c: acid+Mukaiyama reagent,⁵⁴ d: Keckcoupling⁵⁵) led to either decomposition of starting material or aninseparable product mixture of 35 and β,γ-unsaturated ester 36 (Scheme22). The latter presumably arose from acylation of 34 with thevinylketene derived from 6 upon activation of the acyl group. Attemptsto isomerize the C3-C4 double bond of 36 back into conjugation resultedin loss of the carboxylic acid fragment, apparently through aβ-elimination pathway.

With RCM precursor 35 now available, we were positioned to investigatethe cyclization reaction (Scheme 23). In the event, the ring-closingmetathesis conditions employed in our model system (Scheme 18) alsosufficed nicely for the case at hand, delivering macrolactone 37 in ahighly (E)-selective fashion in 69% yield. This corresponds to anincrease in yield by almost 20% compared to our model studies! Finally,removal of the TBS protecting group by buffered hydrogen fluoridecompleted the total synthesis of (+)-migrastatin (1), whose physicaldata (NMR, MS, optical rotation) matched those of migrastatin isolatedfrom natural sources.

Design, Chemical Synthesis, and Evaluation of Migrastatin Analogs.Having achieved our initial objective, a total synthesis of migrastatin,we could take full advantage of our flexible multi-component synthesisfor the subsequent preparation of a variety of analogs. As will beevident, our modular approach served as an excellent platform from whichto quickly explore the SAR profile of migrastatin and assess theanti-metastatic potential of the migrastatin family.

In certain embodiments, our approach with respect to searching for,preparing, and evaluating migrastatin derivatives as to improved cellmigration inhibition properties, comprised three distinct steps: design,chemical synthesis, and biological evaluation. In certain embodiments,the design of migrastatin analogs was aimed at probing the differentregions of migrastatin for their contributions to biological activity.Certain regions of the molecule that we initially considered importantand accessible by synthesis are highlighted in gray below. These regionswere targeted for derivatization.

In certain embodiments, the selection was driven by the followingconsiderations: The glutarimide moiety is a characteristic functionalfeature of migrastatin, and might be indispensable for activity. TheC2-C3 conjugated double bond is a potential site for deactivation by1,4-addition of nucleophiles (e.g. thiols, confer the natural productsNK30424A/B in Scheme 16), or on the contrary, could render migrastatin asuicide inhibitor by covalent bond formation with bionucleophilespresent in the active site of an enzyme. The lactone functionality couldpossibly be a target of hydrolysis in living systems. As such,manipulation of the ester bond might enhance the in vivo stability ofthe molecule. Furthermore, the C6-C12 portion of migrastatin is highlyfunctionalized, and thus, might have biological relevance. One simpleway of exploring this region is through derivatization of the C9hydroxyl group.

The chemical synthesis of migrastatin analogs was accomplished in anefficient manner by utilizing the concept of diverted total synthesis(DTS).²⁸ We put to advantage certain intermediates of the migrastatinsynthesis, such as 26 and 34 (Schemes 20 and 21), as branching points torapidly assemble a chemically diverse set of migrastatin derivatives. Inkeeping with the unique capabilities of diverted total synthesis, wefocused on target structures that would not have been accessible throughmanipulations of the natural product itself or through biosyntheticpathways.

The biological evaluation of the compounds (in terms of their ability toinhibit cell migration) was accomplished in a Boyden chamber cellmigration assay. In this assay, mouse breast tumor cells (4T1 cells) orendothelial cells (HUVECs) are seeded on the upper chamber of atranswell insert. Growth factor-containing serum is added to the lowerchamber. After incubation for 6-8 hours in the presence of differentconcentrations of our analogs, cells that migrated from the upperchamber through the membrane to the lower compartment are counted.Additionally, some of the more potent compounds were tested for theireffect on cell proliferation and metabolic stability in mouse plasma.The study helped provide a broad SAR picture with respect to migrastatinanalogs.

In certain embodiments, synthetic studies towards the preparation ofchemically diversified migrastatin analogs commenced with the synthesisof 2,3-dihydromigrastatin 41 and N-methylated 2,3-dihydromigrastatin 42.Secondary alcohol 34 (Scheme 21), an advanced intermediate involved inthe exemplary migrastatin synthesis described herein, was acylated with6-heptenoyl chloride 38 to deliver RCM precursor 39 (Scheme 24). Asexpected, acylation proceeded smoothly, without the use of the reactionconditions utilized for the acylation of 34 with 2,6-heptadienoic acid 6(Scheme 22). Compound 39 was cyclized to macrolactone 40 by a veryefficient (E)-selective RCM. Cleavage of the TBS ether with HF•pyridineyielded our first analog, 2,3-dihydromigrastatin 41. Alternatively,compound 41 was prepared directly from migrastatin by regioselectivereduction using the Stryker reagent (Scheme 24). The yield of the directtransformation, however, was compromised by the formation of a sideproduct that arose from an intramolecular aldol addition of thetransient copper enolate to the C15 ketone. Methylation of theglutarimide nitrogen was accomplished by treatment of 41 with MeI andCs₂CO₃ in acetone, delivering methylated 2,3-dihydromigrastatin 42 inexcellent yield.

The first set of compounds—migrastatin, together with its analogs 41 and42—was then evaluated in the chamber cell migration assay. The IC₅₀value for fully synthetic migrastatin with 4T1 tumor cells was 29 μM(Table 4); this result was in excellent agreement with that reported byImoto for migrastatin obtained from natural sources. Interestingly,reduction of the C2-C3 double bond and methylation of the glutarimidenitrogen were well tolerated with respect to maintenance of activity.Analogs 41 and 42 are actually slightly more potent than migrastatinitself, with IC₅₀ values of 10 μM and 7 μM, respectively (Table 4).

The small change in inhibitory activity upon alkylation of theglutarimide moiety encouraged us to undertake a more drastic structuralmodification of the migrastatin skeleton. Toward this end, analogs weresynthesized lacking the entire glutarimide-containing side chain, namelymigrastatin core 45 and the corresponding reduced version 48 (Scheme25). Starting from advanced intermediate 26 (Scheme 20), derivatives 45and 48 were quickly assembled via the already establishedacylation-RCM-deprotection sequence. While the reaction of 26 with2,6-heptadienoic acid 6 produced acylated product 43 in only moderate(48%) yield, the acylation steps in the ‘dihydro series’ occurredsmoothly, affording 46 in 82% yield. The same trend was observed for thesubsequent transformation, in which the ring closure was achieved inexcellent (76%) yield for the saturated case (46→47). By contrast, theunsaturated core was delivered in lower (55%) yield (43→44). Finally,protecting group removal delivered macrolactones 45 and 48 withoutcomplications.

In other embodiments, scale-up studies were carried out to establish theapplicability of the exemplary synthesis described herein to thepreparation of migrastatin and analogs thereof in sufficient quantitiesfor biological evaluation in animal models. For example, variation ofthe RCM parameters for a potential large scale preparation of themacrocycles were evaluated. The original reaction conditions called for20 mol % catalyst at 0.5 mM concentration, but as illustrated for thecyclization of 46 to 47 (Scheme 26), the RCM product could be obtainedin just slightly reduced yield by conducting the reaction with 10 mol %catalyst at 5 mM concentration.

Upon examination of compounds 45 and 48 in the cell migration assay, weachieved, to our great surprise, a major breakthrough in potency (Table4). The IC₅₀ values for migrastatin core 45 and macrolactone 48 werefound to be 22 nM and 24 nM, respectively. This translates into anincrease in activity by three orders of magnitude compared tomigrastatin! This appears to lead to the conclusion that the migrastatinside chain may not be required for in vitro inhibition of tumor cellmigration. However, it remains to be determined if migrastatin and coreanalogs 45 and 48 are indeed directed at the same cellular targets.

Another potential interesting site of derivatization of the migrastatinstructure is the C6-C12 region with its two double bonds, threestereocenters, and two heteroatoms. An easy way of derivatizing thisportion of the molecule was found to be the acylation or oxidation ofthe C9 hydroxyl group, producing macrolactones 49 and 50, respectively(Scheme 27). As shown in Table 4, the inhibitory activity of analogs 49and 50 was reduced by roughly an order of magnitude, compared tomacrolactone 48, indicating that the C9 position is sensitive towardmodification.

Starting from our lead compound migrastatin, we were able to reachsimplified congeners/analogs with drastically improved inhibitoryactivities, in particular macrolactones 45 and 48. In anticipation ofsubsequent in vivo evaluation of these compounds and others, preliminarymetabolic stability studies were carried out. Without wishing to bebound to any particular theory, based on our experiences from theepothilone program,⁵⁶ we propose that the ester bond of themacrolactones may be susceptible to ring opening by esterases in mouse(or human) plasma. Such hydrolysis would presumably lead to a loss incompound activity. Accordingly, mouse blood plasma stability ofmigrastatin and novel analogs 41, 42, 45, and 48 was evaluated. Assummarized in Table 5, migrastatin and side chain-containing derivatives41 and 42 were completely inert toward lactone opening over the fulltest period (one hour). However, the most active compounds,macrolactones 45 and 48, were hydrolyzed rapidly (Table 5). Thesefindings are not entirely surprising, considering that the ester bondsin 45 and 48 are sterically less congested relative to those in theother analogs. As a test of the ‘deactivation hypothesis’, thehydrolysis product of macrolactone 48 was prepared (Scheme 27) andtested for its activity against tumor cell migration. Surprisingly,compound 51 was not completely inactive in the chamber assay, butretained a good part of its activity (IC₅₀ value of 378 nM). Therefore,compound 48 might be effective in the projected in vivo models despiteits sensitivity toward hydrolysis. Nevertheless, the data on theunsatisfactory metabolic stability of 45 and 48 influenced us, when weentered the second phase of our analog program. The aspiration ofreaching migrastatin congeners and/or analogs with enhanced plasmastability and retained or improved activity (compared to 45 or 48) ledto the diverted total synthesis of macrolactam 55, macroketone 60(Scheme 28), and the sterically hindered macrolactones 65 and 68 (Scheme29).

The synthesis of analogs 55, 60, 65, and 68 diverged from the originalroute to migrastatin at the stage of the advanced intermediate 26. Forthe preparation of lactam 55, alcohol 26 was subjected to Mitsunobuconditions with DPPA affording allylic azide 52 in 87% yield (Scheme28).⁵⁷ To avoid double bond isomerization of the (Z)-allylic system,azide 52 was immediately reduced following the Staudinger protocol⁵⁷ andsubsequently joined with 6-heptenoic acid under standard peptidecoupling conditions. The resulting product, amide 53, was treated withRCM catalyst 16 under our established reaction conditions, deliveringlactam 54 in 60% yield. The latter was then deprotected with HF•pyridineto afford lactam 55.

The preparation of ketone 60 required the conversion of alcohol 26 intoallylic bromide 56, which was displaced by β-ketosulfone 57 (Scheme28).⁵⁷ Subsequent reductive removal of the sulfone group yielded RCMprecursor 58. The ring closure of 58 to the carbocycle was accomplished,again, very efficiently and selectively by RCM. The desired macroketone60 was obtained following deprotection of 59.

The synthesis of isopropyl macrolactones 65 and 68 also commenced fromalcohol 26 (Scheme 29). Oxidation of 26 generated the corresponding(Z)-enal which was then treated with i-PrMgC1. When the nucleophilicaddition was carried out in THF, an equimolar mixture of the desiredaddition products 61/62 (3:2 ratio) and the reduced product 26 wasobtained. It is well documented in the literature that addition ofisopropyl-Grignard reagents to hindered substrates competes withreduction through hydride delivery from the nucleophile.⁵⁸ Fortunately,the product ratio could be improved by changing the solvent from THF toEt₂O. The reduction pathway was almost completely suppressed by slowaddition of i-PrMgCl to a solution of the aldehyde in Et₂O, whilecarefully maintaining the reaction temperature at −78° C. for severalhours. Diastereomers 61 and 62 were derivatized as their (S)-MPA and(R)-MPA esters (MPA=α-methoxyphenylacetic acid) and analyzed by NMR,leading to the assignment of the newly created stereocenter:⁵⁹ Majorisomer 61 has the ‘unnatural’ (S)-configuration and minor isomer 62 hasthe ‘natural’ (R)-configuration. In addition, the results of the NMRexperiment were probed by a degradation study. We were able to transformcompound 31 (Scheme 21), a synthetic intermediate of the total synthesisof migrastatin, into minor isomer 62, thereby delivering convincingproof for the correctness of the configurational assignment. Thetransformation was accomplished by converting alcohol 31 into itstosylate, reducing the tosylate with LiAlH_(4,) ⁶⁰ and removing the TESprotecting group. For the preparation of lactones 65 and 68, theaddition products 61 and 62 were separated and independently acylated(Scheme 29). Intermediates 63 and 66 were then subjected to our RCMconditions, furnishing the macrocycles 64 and 67 in very good yield.Deprotection occurred smoothly and provided the diastereomeric isopropyllactones 65 and 68.

Indeed, lateral modification of the vulnerable ester bond ofmacrolactones 45 and 48 for more robust entities led to the desiredeffect of enhanced metabolic stability in all four cases: Macrolactam55, macroketone 60, and isopropyl macrolactones 65 and 68 display nosign of degradation in our assay (Table 5). When tested for theirability to inhibit 4T1 cell migration, compounds 55 and 60 were found tobe considerably more active than the natural product migrastatin (255 nMand 100 nM, respectively, Table 4), although some loss of potencyrelative to lactones 45 and 48 was recorded. Surprisingly, incorporationof an isopropyl group at C13 proved to be deleterious for biologicalfunction. Isopropyl macrolides 65 and 68 exhibited only very weakeffects on tumor cell migration (Table 4).

As depicted in Scheme 30, our SAR studies were further diverted on thebasis of macroketone 60. The ketone functionality proved to be anattractive handle for additional derivatization. We started ourexplorations by adding various nucleophiles to the carbonylfunctionality accessing analogs 69-71. Simple NaBH₄ reduction of 60afforded secondary alcohol 69 as a mixture of diastereomers, whileaddition of MeMgBr gave the corresponding tertiary carbinol mixture 70.Following a procedure by Olah,⁶¹ nucleophilic addition of atrifluoromethyl group to 60 was accomplished using(trifluoromethyl)trimethylsilane (TMSCF₃) and catalytic amounts of Bu₄NF(TBAF). This treatment produced the TMS-protected alcohol intermediatewhich was transformed into 71 upon prolonged exposure to TBAF (compound71 was isolated as a single diastereomer after chromatography).Traditional functionalities, such as in oxime 72, could also be easilyincorporated starting from macroketone 60. As a part of our long termgoal of elucidating the cellular target of migrastatin and our newmigrastatin scaffolds, we condensed commercially availableBiotin-dPEG₄™-hydrazide with ketone 60 furnishing the biotin-labeledacyl-hydrazone 73.

Derivatives 69-73 were evaluated for their ability to inhibit tumor cellmigration in the chamber assay. Interestingly, substitution of theketone functionality for a more polar group, such as an alcohol or oximefunction, seems to be detrimental to compound activity. Secondaryalcohol 69, tertiary alcohol 70, and oxime 72 are rather weak migrationinhibitors, with IC₅₀ values of 8.9 μM, 3.1 μM, and 2.3 μM, respectively(Table 4). It appears that incorporation of a trifluoromethyl group cancompensate for the loss of activity caused by the hydroxyl group:Macrocyclic CF₃-alcohol 71 displays the same activity profile asmacroketone 60. Gratifyingly, inhibitory potency is largely retained inbiotinylated hydrazone 73. Therefore, system 73, although a mixture ofgeometric isomers, could qualify as a probe to assist in the targetidentification process.

As discussed above, there are other recently discovered natural productsthat are reported to be strong cell migration inhibitors. In particular,two compounds, epoxyquinol A,¹² a pentaketide dimer with anti-angiogenicactivity, and evodiamine,¹⁵ a potent anti-metastatic and anti-invasivealkaloid, attracted great interest and are currently under seriousinvestigation by several research groups.

These natural products were tested side by side with the inventivemigrastatin analogs for the purpose of validating and calibrating ourassay. As shown in Table 4, the inventive macrolactones outperformevodiamine and are comparable to epoxyquinol A in the chamber assay.

TABLE 4 Chamber Cell Migration Assay with 4T1 Tumor Cells compound IC₅₀(4T1 tumor cells)¹ migrastatin (1)   29 μM 2,3-dihydromigrastatin (41)  10 μM N-methyl-2,3-dihydromigrastatin (42)  7.0 nM migrastatin core(45)   22 nM macrolactone (48)   24 nM acetylated macrolactone (49)  192nM oxidized macrolactone (50)  223 nM hydrolyzed core (51)  378 nMmacrolactam (55)  255 nM macroketone (60)  100 nM (S)-isopropylmacrolactone (65)  227 μM (R)-isopropyl macrolactone (68)  146 μMmacrocyclic secondary alcohol (69)  8.9 μM macrocyclic tertiary alcohol(70)  3.1 μM macrocyclic CF₃-alcohol (71)  101 nM macrooxime (72)  2.3μM biotinylated macrohydrazone (73)  331 nM epoxyquinol   26 nMevodiamine  315 nM ¹Average of three experiments. Each experimentconsists of nine data points (nine different concentrations).

TABLE 5 Metabolic Stability of Selected Compounds in Mouse Plasmacompound stability (t_(1/2), mouse plasma) migrastatin (1) stable¹2,3-dihydromigrastatin (41) stable¹ N-methyl-2,3-dihydromigrastatin (42)stable¹ migrastatin core (45) 20 min macrolactone (48) <5 minmacrolactam (55) stable¹ macroketone (60) stable¹ (S)-isopropylmacrolactone (65) stable¹ (R)-isopropyl macrolactone (68) stable¹¹Intensity of HPLC signal unchanged over 60 min of incubation.

Due to the significance of endothelial cell migration in theangiogenesis process, the chamber cell migration assay described abovewas also conducted with HUVECs (human umbilical vein endothelial cells)and used for the evaluation of our most potent analogs, macrolactones 45and 48, macrolactam 55, and macroketone 60, together with migrastatin asa reference. The IC₅₀ values obtained from this study are listed inTable 6. The general trend in activity, with the simplified analogs 45,48, 55, and 60 being significantly more active against tumor cellmigration than the parent natural product, was also observed forendothelial cells. However, some erosion of potency in the HUVECdetermination compared to the 4T1 cell determination was observed forall compounds tested.

TABLE 6 Chamber Cell Migration Assay with Human Endothelial Cells(HUVECs) compound IC₅₀ (HUVEC)¹ migrastatin (1)  65 μM migrastatin core(45) 150 nM macrolactone (48) 125 nM macrolactam (55)  18 μM macroketone(60)  12 μM ¹Average of three experiments. Each experiment consists ofnine data points (nine different concentrations).

In order to complete the in vitro assay data set for the inventiveanalogs, the effect of migrastatin and cell migration inhibitors 48, 55,and 60 on 4T1 cell proliferation was examined Macrolactone 48,macrolactam 55, and macroketone 60 did not have any cytotoxic oranti-proliferative effects up to 20 μM, whereas migrastatin turned outto be a weak proliferation inhibitor (IC₅₀ value of 42 μM). Withoutwishing to be bound to any particular theory, this outcome appear tolead to the conclusion that cell proliferation inhibition is not acontributor to the effects observed in the chamber assays, and that themigrastatin analogs of the invention may be specific for cell migrationinhibition.

Without wishing to be bound to any particular theory, the followingpreliminary structure-activity relationship (SAR) trends appear toemerge: reduction of the 2,3-double bond of migrastatin results in nosignificant loss of activity. Similarly, alkylation of the glutarimidenitrogen does not appear to negatively impact activity. Complete removalof the C-13 side-chain (e.g., compounds 45, 48, 49, 50, 55, 60 and 71),thereby producing simple macrolactones, dramatically increases activity.This region appears to be relatively sensitive, as indicated by thefollowing observations: replacing the sidechain with a small (isopropyl)mimic results in almost complete loss of activity. When the oxygen ofthe macrolactone is replaced with either a nitrogen or a carbon atom,the effect is much more subtle (activity decreases by about one order ofmagnitude). When the conjugated 2,3-olefin is reduced, activity does notappear to be negatively impacted. For compounds of formula (I) where X₁is CH₂ and Y₁, Y₂ taken together with the carbon atom to which they areattached is C(═O) (i.e., macroketone), oxime formation, reduction oraddition of small nucleophiles to the ketone moiety appears to bedetrimental to activity while the addition of larger nucleophiles (CF₃)is tolerated. The activity of compounds of formula (I) where R₄ is C═Oor OAc (e.g., compounds 49 and 50) is minimally affected (about oneorder of magnitude) as compared to the corresponding compounds where R₄is OH (e.g., compounds 45 and 48).

General Reaction Procedures:

Unless mentioned specifically, reaction mixtures were stirred using amagnetically driven stirrer bar. Reactions involving air ormoisture-sensitive reagents or intermediates were performed under argonor nitrogen atmosphere in glassware which had been heat gun orflame-dried under high vacuum. An inert atmosphere refers to either dryargon or dry nitrogen. Reactions were monitored either by thin layerchromatography, by proton nuclear magnetic resonance (NMR) or byhigh-pressure liquid chromatography (HPLC), of a suitably worked upsample of the reaction mixture.

Indicated reaction temperatures refer to those of the reaction bath,while room temperature (rt) is noted as 22° C. Preparative reactionswere stirred magnetically. Tetrahydrofuran (THF), diethyl ether (Et₂O),methylene chloride (CH₂Cl₂), and toluene were obtained from a drysolvent system (activated alumina columns, positive pressure of argon).All other solvents were used as received in Sure/Seal bottles (Aldrich).Triethylamine (Et₃N), diisopropylethylamine (1-Pr₂NEt), pyridine,2,6-lutidine, and chlorotrimethylsilane (TMSCl) were distilled from CaH₂immediately prior to use. All other reagents were purchased from Aldrichat the highest commercial quality and used without further purification,with the exception of the Stryker reagent which was purchased fromFluka, the RCM catalysts 16 and 17 which were purchased from Strem, andbiotin-dPEG₄-hydrazide which was purchased from Quanta Biodesign.

Listed below are abbreviations used for some common organic reagentsreferred to herein:

CSA: Camphorsulphonic acid

DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene

Dess-Martin: Dess-Martin periodinane

DIBAL-H: Diisobutyl aluminum hydride

DMAP: N,N-Dimethylaminopyridine

DMF: N,N-Dimethylformamide

TBSOTf: Tert-butyl-dimethylsilyl triflate

TESCl: Triethylsilyl chloride

TFA: Trifluoroacetic acid

TMSCl: Trimethylsilyl chloride

THF: Tetrahydrofuran

General Work Up Procedures:

Unless mentioned specifically, reaction mixtures were cooled to roomtemperature or below then quenched, when necessary, with either water ora saturated aqueous solution of ammonium chloride. Desired products wereextracted by partitioning between water and a suitable water-immisciblesolvent (e.g. ethyl acetate, dichloromethane, diethyl ether). Thedesired product containing extracts were washed appropriately with waterfollowed by a saturated solution of brine. On occasions where theproduct containing extract was deemed to contain residual oxidants, theextract was washed with a 10% solution of sodium sulphite in saturatedaqueous sodium bicarbonate solution, prior to the aforementioned washingprocedure. On occasions where the product containing extract was deemedto contain residual acids, the extract was washed with saturated aqueoussodium bicarbonate solution, prior to the aforementioned washingprocedure (except in those cases where the desired product itself hadacidic character). On occasions where the product containing extract wasdeemed to contain residual bases, the extract was washed with 10%aqueous citric acid solution, prior to the aforementioned washingprocedure (except in those cases where the desired product itself hadbasic character). Post washing, the desired product containing extractswere dried over anhydrous magnesium sulphate, and then filtered. Thecrude products were then isolated by removal of solvent(s) by rotaryevaporation under reduced pressure, at an appropriate temperature(generally less than 45° C.).

General Purification Procedures:

Unless mentioned specifically, chromatographic purification refers toflash column chromatography on silica, using a single solvent or mixedsolvent as eluent. Suitably purified desired product containing eluteswere combined and concentrated under reduced pressure at an appropriatetemperature (generally less than 45° C.) to constant mass. Finalcompounds were dissolved in 50% aqueous acetonitrile, filtered andtransferred to vials, then freeze-dried under high vacuum beforesubmission for biological testing.

Analytical Equipment:

Optical rotations were measured on a JASCO DIP-370 digital polarimeterat rt. Concentration (c) in g/100 ml and solvent are given inparentheses. Infrared spectra were obtained on a Perkin-Elmer 1600 FT-IRspectrophotometer neat or as a film in CHCl₃ (NaCl plates). Absorptionbands are noted in cm⁻¹. ¹H- and ¹³C-NMR spectra were recorded on aBruker AMX-400 or a Bruker DRX-500 spectrometer in CDCl₃. Chemicalshifts (δ-values) are reported in ppm with residual undeuterated CHCl₃as the internal standard (referenced to 7.26 ppm for ¹H-NMR and 77.0 ppmfor ¹³C-NMR). Coupling constants (J) (H,H) are given in Hz, spectralsplitting patterns are designated as singulet (s), doublet (d), triplet(t), quadruplet (q), multiplet or more overlapping signals (m), apparent(app), broad signal (br). Low resolution mass spectra (ionspray, avariation of electrospray) were acquired on a Perkin-Elmer Sciex API 100spectrometer. Samples were introduced by direct infusion. Highresolution mass spectra (fast atom bombardment, FAB) were acquired on aMicromass 70-SE-4F spectrometer. Flash chromatography (FC) was performedwith E. Merck silica gel (60, particle size 0.040-0.063 mm). Preparativethin layer chromatography (TLC) was performed with Whatman PartisilPlates (10×10 cm, 60 Å, 200 μm).

Example 1

Vinyl Carbinol 19: Compound 19 was prepared using a slightly modifiedliterature procedure by Madsen. (See, Jorgensen, M.; Iversen, E. H.;Paulsen, A. L.; Madsen, R. J. Org. Chem. 2001, 66, 4630).

Preparation of the Divinylzinc Reagent: to Vinylmagnesium Bromide (294mL, 294 mmol, 1.0M in THF) was added slowly a solution of anhydrousZnCl₂ (20.0 g, 147 mmol, beads) in THF (100 mL) to yield a dark brownsolution of divinylzinc in THF (with some precipitate).

Preparation of vinyl carbinol 19: To a solution of dimethyl2,3-O-isopropylidene-L-tartrate 18 (8.58 g, 39.3 mmol) in toluene (100mL) at −78° C. was added slowly DIBALH (90 mL, 90.0 mmol, 1.0M intoluene). The reaction mixture turned into a white slurry during thecourse of the addition. After stirring for 3 h (the reaction temperaturehas to be kept at −78° C. to prevent overreduction), the divinylzincsolution as prepared above was added to the reaction mixture via cannulaover 45 min. After stirring for another 30 min, the reaction mixture waswarmed to rt and stirred for 4 h. The reaction mixture was thencarefully (!) treated with saturated aqueous NH₄Cl solution and 20%aqueous Na/K-tartrate solution. The organic layer was separated and theaqueous layer was extracted with Et₂O (3×). The combined organic layerswere dried (MgSO₄) and concentrated under reduced pressure. Purificationof the crude product by FC (hexane/EtOAc 4:1) afforded vinyl carbinol 19(6.28 g, 75%, diastereoselectivity >90%) as a colorless oil. ¹H-NMR (400MHz, CDCl₃) g 6.04-5.94 (m, 2H), 5.40 (d, J=17.3, 2H), 5.30 (d, J=10.5,2H), 4.19-4.16 (m, 2H), 3.89-3.87 (m, 2H), 2.91 (br s, 2H), 1.42 (s,6H).

Example 2

1,2-Diol 20: The preparation of compound 20 has been reported before byChang, (See, Lee, W. W.; Chang, S. Tetrahedron: Asymmetry 1999, 10,4473) but experimental details have not been provided.

To a solution of vinyl carbinol 19 (6.28 g, 29.2 mmol) in DMF (100 mL)at 0° C. was added NaH (2.58 g, 64.5 mmol, 60% dispersion in mineraloil) and, 5 min later, MeI (4.38 mL, 70.3 mmol). The reaction mixturewas warmed to rt, stirred for 45 min, and then treated with 2M NH₄OH.The organic layer was separated and the aqueous layer was extracted withEt₂O (3×). The combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure. The crude product was dissolved inMeOH (150 mL) and 2M HCl (50 mL) and heated to reflux for 2 h. Thereaction mixture was cooled to rt, treated with saturated aqueous Na₂CO₃solution and diluted with Et₂O. The organic layer was separated and theaqueous layer was extracted with Et₂O (3×). The combined organic layerswere dried (MgSO₄) and concentrated under reduced pressure. Purificationof the crude product by FC (hexane/EtOAc 2:1) afforded 1,2-diol 20 (4.72g, 80%) as a colorless oil. [α]_(D) +31.0° (c 1.77, CHCl₃); IR (neat)3454, 3078, 2982, 2936, 2824, 1643, 1420, 1192, 1102, 992; ¹H-NMR (500MHz, CDCl₃) δ 5.77-5.71 (m, 2H), 5.36-5.32 (m, 4H), 3.81 (app t, J=6.3,2H), 3.76 (d, J=5.5, 2H), 3.32 (s, 6H), 2.96 (br s, 2H); ¹³C-NMR (125MHz, CDCl₃) δ 135.08, 119.27, 86.65, 71.23, 57.15; MS (ESI) 225 [M+Na⁺];HRMS (FAB) calcd. for C₁₀H₁₈O₄Na [M+Na⁺] 225.1103, found 225.1079.

Example 3

Butadiene 3: Compound 3 was prepared using modified literatureprocedures (See, Danishefsky, S. J. et al.; J. J. Am. Chem. Soc. 1979,101, 7001).

To a suspension of NaH (4.40 g, 110 mmol, 60% dispersion in mineral oil)in toluene (90 mL) and MeOH (0.1 mL) at 0° C. was added a mixture of3-pentanone (10.6 mL, 105 mmol) and methyl formate (8.00 mL, 130 mmol)over 1 hr. The reaction mixture was warmed to rt, stirred for another 3h, and then diluted with Et₂O. The suspension was filtered and theprecipitate was washed with Et₂O. The resulting crude sodium salt of1-hydroxy-2-methyl-1-penten-3-one was dissolved in DMSO (100 mL) andMe₂SO₄ (9.16 mL, 97.0 mmol) was added at rt. After stirring for 30 min,the reaction mixture was treated with 2M NH₄OH and diluted with Et₂O.The organic layer was separated, washed with H₂O and saturated aqueousNaCl solution, dried (MgSO₄), and concentrated under reduced pressure toafford 1-methoxy-2-methyl-1-penten-3-one (8.27 g, 74%). To a solution of1-methoxy-2-methyl-1-penten-3-one (2.60 g, 20.3 mmol) in Et₂O (12.0 mL)was added Et₃N (7.08 mL, 50.8 mmol) and TMSOTf (3.68 mL, 20.3 mL) at 0°C. The reaction mixture was warmed to rt, stirred for another 3 h, andthen poured onto a saturated aqueous NaHCO₃ solution. The organic layerwas separated, washed with saturated aqueous NaCl solution, dried(MgSO₄), and concentrated under reduced pressure to afford butadiene 3(3.66 g, 90%). ¹H-NMR (400 MHz, CDCl₃) δ 6.35 (s, 1H), 4.75 (q, J=6.9,1H), 3.63 (s, 3H), 1.66 (s, 3H), 1.62 (d, J=6.9, 3H), 0.22 (s, 9H).

Example 4

Dihydropyrone 21: To a solution of diol 20 (2.55 g, 12.6 mmol) in CH₂Cl₂(130 mL) at 0° C. was added Na₂CO₃ (1.40 g, 13.2 mmol) and Pb(OAc)₄(5.87 g, 13.2 mmol). The reaction mixture was warmed to rt, stirred for25 min, and then treated with ethylene glycol (300 μL). After stirringfor another 5 min, the reaction mixture was filtered through a Celitepad. The filtrate was washed with saturated aqueous NaHCO₃ solution andsaturated aqueous NaCl solution and dried (MgSO₄). The obtained solutionof α-methoxy-α-vinyl aldehyde 2 in CH₂Cl₂ was cooled to −78° C., andthen TiCl₄ (2.77 mL, 25.2 mmol) and butadiene 3 (6.06 g, 30.3 mmol) wereadded. After stirring for 20 min, the reaction mixture was treated withMeOH (5 min), followed by the addition of saturated aqueous NaHCO₃solution and 20% aqueous Na/K-tartrate solution. The organic layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (3×). Thecombined organic layers were dried (MgSO₄) and concentrated underreduced pressure. The crude product was dissolved in CH₂Cl₂ (130 mL) andTFA (13 mL) and stirred for 1 hr. Toluene (50 mL) was added and thereaction mixture was concentrated under reduced pressure. Purificationof the crude product by FC (hexane/EtOAc 20:1→10:1→7:1) affordeddihydropyrone 21 (4.31 g, 87%) as a colorless oil. [α]_(D) +77.1° (c2.00, CHCl₃); IR (neat) 2980, 2938, 2883, 2827, 1785, 1671, 1622, 1602,1460, 1387, 1305, 1214, 1176, 1085, 1010; ¹H-NMR (500 MHz, CDCl₃) δ 7.36(s, 1H), 5.63-5.54 (m, 1H), 5.48-5.43 (m, 2H), 4.25 (dd, J=8.6, 2.9,1H), 3.88 (app t, J=8.5, 1H), 3.37 (s, 3H), 2.44 (dq, J=7.2, 2.9, 1H),1.68 (s, 3H), 1.07 (d, J=7.2, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 198.99,160.75, 131.79, 122.06, 112.51, 82.69, 81.99, 56.37, 40.62, 10.42, 9.96;MS (ESI) 219 [M+Na⁺]; HRMS (FAB) calcd. for C₁₁H₁₆O₃Na [M+Na⁺] 219.0997,found 219.0991.

Example 5

Diol 25: To a solution of dihydropyrone 21 (4.30 g, 219 mmol) in THF (50mL) at −10° C. was added MeOH (977 μL, 24.1 mmol) and LiBH₄ (12.1 mL,24.1 mmol, 2M in THF). After stirring for 10 min, the reaction mixturewas carefully treated with 0.2M HCl (25 mL) and stirring was continuedfor another 20 min. Then the organic layer was separated and the aqueouslayer was extracted with EtOAc (4×). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure. The crude alcohol22 was dissolved in THF (280 mL) and H₂O (28 mL), and champhorsulfonicacid (1.02 g, 4.38 mmol) was added. After refluxing for 2 h, thereaction mixture was treated with saturated aqueous NaHCO₃ solution. Theorganic layer was separated and the aqueous layer was extracted withEtOAc (3×). The combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure. The crude lactol 23 was dissolvedin THF (60 mL) and H₂O (15 mL), and LiBH₄ (12.1 mL, 24.1 mmol, 2M inTHF) was added at rt. After stirring for 15 min, the reaction mixturewas treated with 0.2M HCl (35 mL) and stirring was continued for another20 min. Then the organic layer was separated and the aqueous layer wasextracted with EtOAc (3×). The combined organic layers were dried(MgSO₄) and concentrated under reduced pressure. Purification of thecrude product by FC (hexane/EtOAc 4:1→2:1→1:1) afforded diol 25 (2.34 g,53%) as a colorless oil. [α]_(D) +40.0° (c 1.00, CHCl₃); IR (CHCl₃)3621, 3565, 3444, 3012, 2934, 2868, 1449, 1393, 1238, 1083; ¹H-NMR (500MHz, CDCl₃) δ 5.74-5.67 (m, 1H), 5.33-5.25 (m, 2H), 5.16 (d, J=10.2,1H), 4.12 (d, J=11.9, 1H), 3.95 (d, J=11.9, 1H), 3.48 (dd, J=8.1, 5.4,1H), 3.26 (app t, J=5.5, 1H), 3.23 (s, 3H), 2.74-2.68 (m, 1H), 2.57 (brs, 2H), 1.79 (d, J=1.4, 3H), 0.98 (d, J=6.9, 3H); ¹³C-NMR (125 MHz,CDCl₃) δ 135.32, 135.20, 130.41, 119.51, 83.42, 77.44, 61.51, 55.93,34.80, 21.89, 16.85; MS (ESI) 223 [M+Na⁺]; HRMS (FAB) calcd. forC₁₁H₂₀O₃Na [M+Na⁺] 223.1310, found 223.1301.

Example 6

Dimeric Acetal 24: The Ferrier rearrangement described above was carriedout at a concentration of 0.07M. When the Ferrier rearrangement wasconducted at a concentration of 0.30M, the formation of a side product,which corresponds to dimeric acetal 24, was observed. Compound 24 wasisolated after FC (hexane/EtOAc 20:1→10:1) in 15-20% yield as a whitecrystalline solid. M.p. 83-85° C.; [α]_(D) −161.3° (c 1.00, CHCl₃); IR(CHCl₃) 3003, 2910, 2816, 1446, 1382, 1317, 1211, 1088, 965; ¹H-NMR (500MHz, CDCl₃) δ 5.67-5.59 (m, 4H), 5.39-5.28 (m, 6H), 3.93 (dd, J=8.4,2.9, 2H), 3.57 (app t, J=8.2, 2H), 3.32 (s, 6H), 1.94-1.91 (m, 2H), 1.74(s, 6H), 0.91 (d, J=6.8, 6H); ¹³C-NMR (125 MHz, CDCl₃) δ 134.66, 132.01,129.40, 119.11, 93.37, 83.15, 72.19, 56.79, 30.44, 18.81, 12.78; MS(ESI) 401 [M+Na⁺]; HRMS (FAB) calcd. for C₂₂H₃₅O₅ [M+H⁺] 1379.2485,found 379.2486.

Example 7

Monoprotected Diol 26: To a solution of diol 25 (364 mg, 1.82 mmol) inCH₂Cl₂ (8 mL) at rt was added 2,6-lutidine (530 μL, 4.55 mmol) andTBSOTf (961 μL, 4.19 mmol). After stirring for 20 min, the reactionmixture was treated with saturated aqueous NaHCO₃ solution. The organiclayer was separated and the aqueous layer was extracted with CH₂Cl₂(3×). The combined organic layers were dried (MgSO₄) and concentratedunder reduced pressure. Purification of the crude product by FC(hexane/EtOAc 30:1) afforded the corresponding diprotected diol (731 mg,94%) as a colorless oil. [α]_(D) + 0.1° (c 1.00, CHCl₃); IR (CHCl₃)2929, 2856, 1472, 1253, 1076; ¹H-NMR (500 MHz, CDCl₃) δ 5.67-5.60 (m,1H), 5.29-5.21 (m, 3H), 4.14 (d, J=11.8, 1H), 4.04 (d, J=11.8, 1H), 3.43(dd, J=7.2, 2.9, 1H), 3.37 (app t, J=7.5, 1H), 3.21 (s, 3H), 2.60-2.56(m, 1H), 1.72 (d, J=0.9, 3H), 0.91-0.89 (m, 21H), 0.05-0.04 (m, 12H);¹³C-NMR (125 MHz, CDCl₃) δ 135.44, 133.27, 131.41, 118.43, 86.20, 78.68,61.91, 56.17, 33.85, 26.17, 25.93, 20.99, 18.56, 18.36, 14.13, −3.82,−4.80, −5.29; MS (ESI) 451 [M+Na⁺]; HRMS (FAB) calcd. for C₂₃H₄₈O₃Si₂Na[M+Na⁺] 451.3040, found 451.3054.

A solution of the diprotected diol (731 mg, 1.71 mmol) in HOAc (9 mL),THF (3 mL), and H₂O (3 mL) was stirred at rt for 8 h. The reactionmixture was neutralized with solid Na₂CO₃ and diluted with H₂O and Et₂O.The organic layer was separated and the aqueous layer was extracted withEt₂O (3×). The combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure. Purification of the crude productby FC (hexane/EtOAc 10:1→5:1) afforded monoprotected diol 26 (456 mg,85%) as a colorless oil. [α]_(D) +3.8° (c 1.85, CHCl₃); IR (neat) 3352,2957, 2930, 2857, 1472, 1462, 1250, 1127, 1081, 1028; ¹H-NMR (500 MHz,CDCl₃) δ 5.73-5.66 (m, 1H), 5.30-5.24 (m, 3H), 4.12 (dd, J=11.8, 4.9,1H), 4.00 (dd, J=11.8, 6.5, 1H), 3.48-3.43 (m, 2H), 3.22 (s, 3H),2.69-2.61 (m, 1H), 1.78 (d, J=1.1, 3H), 1.68 (br t, 1H), 0.90-0.89 (m,12H), 0.06 (s, 3H), 0.04 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 135.15,133.05, 118.54, 85.89, 78.28, 61.76, 56.12, 34.23, 26.11, 25.64, 21.53,18.49, 15.32, −3.88, −4.70; MS (ESI) 337 [M+Na⁺]; HRMS (FAB) calcd. forC₁₇H₃₄O₃SiNa [M+Na⁺] 337.2175, found 337.2162.

Example 8

Propionyl Oxazolidinone 28: Compound 28 was prepared by reaction of(R)-(+)-4-benzyl-2-oxazolidinone with BuLi and propionyl chloride in THFaccording to standard literature procedures (See, Evans, D. A.Aldrichimica Acta 1982, 15, 23).

Example 9

Aldol Product 29: To a solution of alcohol 26 (189 mg, 0.601 mmol) inCH₂Cl₂ (4 mL) at rt was added Dess-Martin periodinane (280 mg, 0.661mmol). After stirring for 50 min, the reaction mixture was treated withsaturated aqueous Na₂S₂O₃ solution and saturated aqueous NaHCO₃solution. The organic layer was separated and the aqueous layer wasextracted with CH₂Cl₂ (3×). The combined organic layers were dried(MgSO₄) and concentrated under reduced pressure to yield crude aldehyde27. IR (neat) 2958, 2936, 2891, 2858, 1674, 1467, 1378, 1249, 1126,1093, 1031; ¹H-NMR (500 MHz, CDCl₃) δ 10.06 (s, 1H), 6.51 (dd, J=10.7,1.5, 1H), 5.63 (ddd, J=17.4, 10.5, 7.9, 1H), 5.32-5.25 (m, 2H), 3.56(dd, J=6.6, 3.8, 1H), 3.45 (app t, J=7.3, 1H), 3.42-3.35 (m, 1H), 3.20(s, 3H), 1.75 (s, 3H), 1.03 (d, J=6.6, 3H), 0.91 (s, 9H), 0.07 (s, 3H),0.02 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 191.36, 153.37, 134.76, 133.96,119.12, 85.73, 78.03, 56.21, 33.15, 26.05, 18.44, 16.37, 14.73, −3.84,−4.85.

The crude aldehyde 27 was dissolved in EtOAc (2 mL) and added to neatpropionyl oxazolidinone 28 (210 mg, 0.902 mmol). The reaction mixturewas then treated at rt with anhydrous MgCl₂ (57 mg, 0.601 mmol), Et₃N(210 μL, 1.50 mmol), and TMSCl (153 μL, 1.20 mmol). After stirring for36 h, the reaction mixture was filtered through a silica plug (Et₂O) andthe filtrate was concentrated under reduced pressure. The residual oilwas dissolved in MeOH (3 mL), treated with TFA (1 drop) and stirred for10 min. Toluene (3 mL) was added and the reaction mixture wasconcentrated under reduced pressure. Purification of the crude productby FC (hexane/CH₂Cl₂ 1:1→CH₂Cl₂) afforded aldol product 29 (219 mg, 67%)as a colorless oil. [α]_(D) −16.1° (c 1.77, CHCl₃); IR (neat) 3505,2920, 2856, 1782, 1699, 1453, 1384, 1258, 1208, 1125, 1079, 1020; ¹H-NMR(500 MHz, CDCl₃) δ 7.35-7.26 (m, 5H), 5.64 (ddd, J=17.6, 10.3, 7.6, 1H),5.56 (d, J=10.2, 1H), 5.37 (dd, J=10.4, 1.8, 1H), 5.30 (dd, J=17.4, 1.8,1H), 4.73-4.69 (m, 2H), 4.22-4.16 (m, 2H), 4.14-4.08 (m, 1H), 3.46 (dd,J=8.0, 1.8, 1H), 3.39 (app t, J=8.0, 1H), 3.36 (dd, J=14.1, 3.8, 1H),3.21 (s, 3H), 2.81 (dd, J=13.6, 9.6, 1H), 2.75-2.68 (m, 1H), 2.39 (br s,1H), 1.75 (s, 3H), 1.02 (d, J=7.0, 3H), 0.92 (s, 9H), 0.91 (d, J=6.0,3H), 0.07 (s, 3H), 0.04 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 176.48,153.94, 135.68, 135.35, 134.76, 131.41, 129.52, 128.94, 127.29, 119.26,86.43, 78.16, 72.78, 66.06, 56.01, 55.76, 41.09, 37.74, 33.44, 26.16,18.60, 17.16, 14.48, 13.60, −3.74, −4.86; MS (ESI) 546 [M+H⁺]; HRMS(FAB) calcd. for C₃₀H₄₈NO₆Si [M+H⁺] 546.3251, found 546.3251.

Example 10

Primary Alcohol 31: To a solution of aldol product 29 (215 mg, 0.394) inCH₂Cl₂ (5 mL) at rt was added imidazole (107 mg, 1.58 mmol) and TESCl(198 μL, 1.18 mmol). After stirring for 12 h, the reaction mixture wastreated with H₂O and diluted with CH₂Cl₂. The organic layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (3×). Thecombined organic layers were dried (MgSO₄) and concentrated underreduced pressure to afford the TES-protected aldol product 30. The crudeproduct 30 was dissolved in THF (5 mL), and MeOH (64 μL, 0.394 mmol) andLiBH₄ (35 mg, 1.58 mmol) were added at rt. After stirring for 1 h, thereaction mixture was treated with 0.5M NaOH. The organic layer wasseparated and the aqueous layer was extracted with Et₂O (3×). Thecombined organic layers were dried (MgSO₄) and concentrated underreduced pressure. Purification of the crude product by FC (hexane/EtOAc10:1) afforded primary alcohol 31 (159 mg, 83%) as a colorless oil.[α]_(D) +10.9° (c 2.38, CHCl₃); IR (neat) 3460, 2970, 2930, 2880, 1460,1380, 1250, 1130, 1060, 1020; ¹H-NMR (500 MHz, CDCl₃) δ 5.60-5.53 (m,1H), 5.35-5.26 (m, 3H), 4.31 (d, J=9.1, 1H), 3.68-3.58 (m, 2H),3.42-3.34 (m, 2H), 3.20 (s, 3H), 3.13 (app d, J=7.0, 1H), 2.65-2.59 (m,1H), 1.94-1.88 (m, 1H), 1.67 (d, J=1.2, 3H), 0.94 (t, J=8.0, 9H),0.93-0.91 (m, 12H), 0.70 (d, J=7.1, 3H), 0.58 (q, J=8.0, 6H), 0.04 (s,3H), 0.00 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 135.10, 133.66, 133.46,118.84, 86.46, 78.30, 76.58, 68.33, 56.08, 38.87, 33.24, 26.13, 18.58,17.70, 14.25, 12.64, 6.75, 4.74, −3.85, −4.89; MS (ESI) 509 [M+Na⁺];HRMS (FAB) calcd. for C₂₆H₅₄O₄Si₂Na [M+Na⁺] 509.3458, found 509.3468.

Example 11

Glutarimide Aldehyde 5: Compound 5 was synthesized according to aliterature procedure (See, Egawa, Y. et al.; Chem. Pharm. Bull. 1963,11, 589).

Example 12

Enone 33: To a solution of primary alcohol 31 (142 mg, 0.292 mmol) inCH₂Cl₂ (5 mL) at rt was added Dess-Martin periodinane (136 mg, 0.321mmol). After stirring for 45 min, the reaction mixture was treated withsaturated aqueous Na₂S₂O₃ solution and saturated aqueous NaHCO₃solution. The organic layer was separated and the aqueous layer wasextracted with CH₂Cl₂ (3×). The combined organic layers were dried(MgSO₄) and concentrated under reduced pressure. In a separate flask,dimethyl methylphosphonate (316 μL, 2.92 mmol) in THF (2 mL) at −78° C.was treated with BuLi (1.64 mL, 2.62 mmol, 1.6M in hexane). Afterstirring for 20 min, the crude aldehyde obtained from the Dess-Martinoxidation was dissolved in THF (1 mL) and added to the reaction mixture.The reaction mixture was warmed to 0° C., stirred for 15 min, and thentreated with saturated aqueous NH₄Cl solution. The organic layer wasseparated and the aqueous layer was extracted with EtOAc (4×). Thecombined organic layers were dried (MgSO₄) and concentrated underreduced pressure. The crude product was dissolved in CH₂Cl₂ (5 mL), andDess-Martin periodinane (136 mg, 0.321 mmol) was added at rt. Afterstirring for 20 min, the reaction mixture was treated with saturatedaqueous Na₂S₂O₃ solution and saturated aqueous NaHCO₃ solution. Theorganic layer was separated and the aqueous layer was extracted withCH₂Cl₂ (1×) and EtOAc (3×). The combined organic layers were dried(MgSO₄) and concentrated under reduced pressure. The crude phosphonate32 was put under high vacuum for 1 hr. To a solution of the crudeproduct 32 in MeCN (5 mL) at rt was added anhydrous LiCl (25 mg, 0.583mmol) and DBU (87 μL, 0.583 mmol). After stirring for 10 min, a solutionof glutarimide aldehyde 5 (136 mg, 0.875 mmol) in MeCN (1 mL) was added.After stirring for 1 h, the reaction mixture was treated with saturatedaqueous NH₄Cl solution and diluted with EtOAc. The organic layer wasseparated and the aqueous layer was extracted with EtOAc (3×). Thecombined organic layers were dried (MgSO₄) and concentrated underreduced pressure. Purification of the crude product by FC (hexane/EtOAc4:1→2:1) afforded enone 33 (105 mg, 57%) as a colorless oil. [α]_(D)+4.4° (c 1.69, CHCl₃); IR (neat) 2955, 2931, 2877, 2855, 1722, 1698,1628, 1461, 1377, 1288, 1254, 1128, 1066, 1035; ¹H-NMR (500 MHz, CDCl₃)δ 7.91 (br s, 1H), 6.71-6.67 (m, 1H), 6.26 (d, J=15.9, 1H), 5.65 (ddd,J=17.4, 10.4, 8.4, 1H), 5.41-5.36 (m, 2H), 5.29 (dd, J=17.4, 1.6, 1H),4.62 (d, J=9.3, 1H), 3.43 (app d, J=7.2, 1H), 3.38-3.33 (m, 1H), 3.21(s, 3H), 3.09-2.99 (m, 1H), 2.75-2.66 (m, 3H), 2.36-2.28 (m, 5H), 1.66(s, 3H), 0.91 (s, 9H), 0.87-0.82 (m, 15H), 0.46 (q, J=7.9, 6H), 0.05 (s,3H), −0.01 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 202.98, 171.17, 140.21,134.99, 134.43, 134.09, 132.47, 119.18, 86.57, 78.53, 72.86, 55.97,47.67, 37.47, 37.43, 37.27, 33.22, 29.82, 29.69, 26.13, 18.59, 14.18,12.53, 6.76, 4.71, −3.83, −4.91; MS (ESI) 636 [M+H⁺]; HRMS (FAB) calcd.for C₃₄H₆₂NO₆Si₂ [M+H⁺] 636.4116, found 636.4116.

Example 13

Secondary Alcohol 34: To a solution of enone 33 (101 mg, 0.159 mmol) intoluene (4.5 mL) at rt was added the Stryker reagent (156 mg, 0.079mmol, dark red solid if quality is good). After stirring for 3.5 h,hexane (3 mL) was added, and the reaction mixture was exposed to air,stirred for 20 min, and concentrated under reduced pressure.Purification of the crude product by FC (hexane/EtOAc 6:1→2:1) affordedthe corresponding saturated ketone as a colorless oil. [α]_(D) +7.7° (c3.00, CHCl₃); IR (neat) 3217, 2954, 2932, 2877, 1713, 1459, 1377, 1253,1126, 1061, 1035, 1006; ¹H-NMR (500 MHz, CDCl₃) δ 8.05 (br s, 1H), 5.63(ddd, J=17.0, 10.0, 8.2, 1H), 5.40-5.36 (m, 2H), 5.28 (dd, J=17.0, 1.8,1H), 4.55 (d, J=9.4, 1H), 3.41 (dd, J=8.2, 1.2, 1H), 3.35 (app t, J=8.2,1H), 3.19 (s, 3H), 2.82-2.64 (m, 4H), 2.60-2.41 (m, 2H), 2.29-2.23 (m,2H), 2.18-2.10 (m, 1H), 1.62 (d, J=1.2, 3H), 1.61-1.53 (m, 2H),1.43-1.34 (m, 2H), 0.92-0.90 (m, 12H), 0.86 (t, J=7.8, 9H), 0.77 (d,J=7.0, 3H), 0.46 (q, J=7.8, 6H), 0.03 (s, 3H), −0.02 (s, 3H); ¹³C-NMR(125 MHz, CDCl₃) δ 213.45, 172.06, 134.91, 134.57, 132.25, 119.24,86.58, 78.52, 72.90, 55.94, 49.29, 44.53, 37.75, 34.32, 33.18, 30.44,26.11, 19.97, 18.57, 17.16, 13.90, 12.46, 6.75, 4.71, −3.85, −4.93; MS(ESI) 638 [M+H⁺]; HRMS (FAB) calcd. for C₃₄H₆₄NO₆Si₂ [M+H⁺] 638.4272,found 638.4273.

A solution of the saturated ketone in HOAc (3 mL), THF (1 mL), and H₂O(1 mL) was stirred at rt for 2 h. The reaction mixture was neutralizedwith solid Na₂CO₃ and diluted with H₂O and EtOAc. The organic layer wasseparated and the aqueous layer was extracted with EtOAc (3×). Thecombined organic layers were dried (MgSO₄) and concentrated underreduced pressure. Purification of the crude product by FC (hexane/EtOAc4:1→1:1) afforded secondary alcohol 34 (68 mg, 82%) as a white foam.[α]_(D) +1.0° (c 1.00, CHCl₃); IR (CHCl₃) 3601, 3366, 3035, 2931, 2861,1708, 1455, 1378, 1249, 1120, 1026; ¹H-NMR (500 MHz, CDCl₃) δ 8.22 (brs, 1H), 5.63-5.56 (m, 1H), 5.48 (d, J=9.3, 1H), 5.33 (dd, J=10.3, 1.5,1H), 5.27 (dd, J=17.2, 1.5, 1H), 4.60 (d, J=9.8, 1H), 3.42-3.35 (m, 2H),3.18 (s, 3H), 2.79-2.63 (m, 4H), 2.58-2.54 (m, 2H), 2.29-2.23 (m, 2H),2.18-2.10 (m, 1H), 1.95 (br s, 1H), 1.67 (d, J=1.0, 3H), 1.66-1.59 (m,2H), 1.42-1.37 (m, 2H), 0.91 (s, 9H), 0.89 (d, J=6.6, 3H), 0.87 (d,J=7.1, 3H), 0.05 (s, 3H), 0.01 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ214.01, 172.21, 135.51, 134.72, 131.56, 119.19, 86.30, 78.26, 71.69,55.98, 48.87, 42.70, 37.73, 37.70, 34.08, 33.26, 30.32, 26.11, 20.07,18.55, 17.35, 13.87, 13.63, −3.79, −4.90; MS (ESI) 546 [M+Na⁺]; HRMS(FAB) calcd. for C₂₈H₄₉NO₆SiNa [M+Na⁺] 546.3227, found 546.3227.

Example 14

2,6-Heptadienoic Acid 6: Compound 6 can be prepared by γ-alkylation ofcrotonic acid with allyl bromide (See, Katzenellenbogen, J. A. et al.;J. Chem. Soc., Perkin Trans. 1 1998, 2721). However, it was found thatthe procedure described below is more convenient for larger scalepreparations of 2,6-heptadienoic acid 6.

To a solution of oxalyl chloride (3.36 mL, 39.2 mmol) in CH₂Cl₂ (100 mL)at −78° C. was added DMSO (5.56 mL, 78.3 mmol). After stirring for 5min, 4-penten-1-ol (2.00 mL, 19.6 mmol) was added, and after another 15min Et₃N (13.6 mL, 97.9 mmol) was added. The reaction mixture was warmedto rt and then treated with 0.1M HCl. The organic layer was separated,washed with saturated aqueous NaCl solution, dried (MgSO₄), and treatedwith Ph₃PCHCO₂t-Bu (7.38 g, 19.6 mmol) at rt. The reaction mixture wasstirred for 5 h and then treated with saturated aqueous NH₄Cl solutionand diluted with CH₂Cl₂. The organic layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (3×). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure. The crude productwas filtered through a silica plug (CH₂Cl₂/pentane 1:1) to give t-butyl(E)-2,6-heptadienoate. To a solution of this ester in CH₂Cl₂ (40 mL) wasadded TFA (5 mL) at rt. After stirring for 12 h, the reaction mixturewas concentrated under reduced pressure. Purification of the crudeproduct by FC (hexane/EtOAc 15:1→5:1) afforded 2,6-heptadienoic acid 6(1.67 g, 68%) as a colorless oil. ¹H-NMR (400 MHz, CDCl₃) δ 7.12-7.05(m, 1H), 5.88-5.76 (m, 2H), 5.08-5.02 (m, 2H), 2.37-2.32 (m, 2H),2.26-2.21 (m, 2H).

Example 15

Formation of the mixed anhydride: To a solution of 2,6-heptadienoic acid6 (68 mg, 0.535 mmol) in toluene (1 mL) at rt was added2,4,6-trichlorobenzoyl chloride (84 μL, 0.535 mmol) and i-Pr₂NEt (89 μL,0.508 mmol). The reaction mixture was stirred for 3 h and then used asit is as a stock solution (0.54M) for the subsequent acylationreactions.

Example 16

Unsaturated Ester 35: To a solution of alcohol 34 (41 mg, 0.078 mmol) intoluene (0.1 mL) at rt was added pyridine (25 μL, 0.313 mmol) and themixed anhydride (See above for the preparation of a stock solution ofthe mixed anhydride in toluene) (460 μL, 0.235 mmol, 0.54M in toluene).After stirring for 24 h, the reaction mixture was directly loaded onto asilica column and purified by FC (hexane/EtOAc 10:1→4:1→2:1) to affordunsaturated ester 35 (33 mg, 67%) as a colorless oil. [α]_(D) −29.0° (c1.00, CHCl₃); IR (neat) 3214, 3081, 2930, 2856, 1722, 1452, 1377, 1256,1126, 1028; ¹H-NMR (500 MHz, CDCl₃) δ 7.87 (br s, 1H), 6.89 (app dt,J=15.5, 6.8, 1H), 5.81-5.62 (m, 4H), 5.61 (dd, J=10.3, 1.2, 1H), 5.38(dd, J=10.3, 1.8, 1H), 5.32 (dd, J=17.3, 1.4, 1H), 5.03-4.98 (m, 2H),3.43-3.39 (m, 2H), 3.21 (s, 3H), 3.00-2.85 (m, 2H), 2.72-2.68 (m, 2H),2.56-2.44 (m, 2H), 2.30-2.24 (m, 4H), 2.23-2.08 (m, 3H), 1.62 (s, 3H),1.61-1.58 (m, 2H), 1.36-1.32 (m, 2H), 0.94 (app t, J=7.2, 6H), 0.90 (s,9H), 0.06 (s, 3H), 0.00 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 211.20,171.83, 164.68, 148.92, 137.72, 136.96, 134.57, 127.00, 121.15, 119.23,115.60, 86.28, 78.39, 73.79, 56.03, 47.39, 41.45, 37.72, 34.16, 33.84,31.97, 31.46, 30.40, 26.21, 26.13, 20.10, 18.59, 17.70, 13.72, 12.66,−3.76, −4.94; MS (ESI) 654 [M+Na⁺]; HRMS (FAB) calcd. for C₃₅H₅₇NO₇SiNa[M+Na⁺] 654.3826, found 654.3835.

Example 17

TBS-Migrastatin 37: To a solution of unsaturated ester 35 (29 mg, 0.046mmol) in refluxing toluene (100 mL) was added Grubbs-II catalyst 16 (8mg, 0.0092 mmol). After stirring for 15 min, the reaction mixture wascooled to rt and filtered through a silica plug (hexane/EtOAc 1:3).Purification of the crude product by FC (hexane/EtOAc 5:1→2:1→1:1)afforded TBS-migrastatin 37 (19 mg, 69%) as a white solid. [α]_(D)+13.7° (c 0.50, CHCl₃); ¹H-NMR (500 MHz, CDCl₃) δ 7.77 (br s, 1H),6.54-6.48 (m, 1H), 5.59 (d, J=15.7, 1H), 5.56 (d, J=10.7, 1H), 5.51-5.45(m, 1H), 5.22 (dd, J=15.4, 4.6, 1H), 5.08 (d, J=9.5, 1H), 3.39 (dd,J=8.1, 4.6, 1H), 3.19 (s, 3H), 3.03 (app d, J=7.8, 1H), 2.98-2.92 (m,1H), 2.91-2.85 (m, 1H), 2.73-2.68 (m, 2H), 2.50 (app t, J=6.9, 2H),2.44-2.40 (m, 2H), 2.29-2.09 (m, 5H), 1.81 (d, J=1.1, 3H), 1.64-1.57 (m,2H), 1.37-1.31 (m, 2H), 1.11 (d, J=7.2, 3H), 0.92-0.90 (m, 12H), 0.04(s, 3H), −0.01 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 210.81, 171.82,163.80, 150.36, 133.94, 130.17, 129.49, 128.78, 121.88, 83.37, 79.25,76.82, 56.68, 51.15, 40.24, 37.70, 37.68, 34.17, 33.47, 31.15, 30.36,30.27, 26.29, 20.12, 18.63, 13.61, 13.30, −3.61, −4.95; MS (ESI) 626[M+Na⁺]; HRMS (FAB) calcd. for C₃₃H₅₃NO₇SiNa [M+Na⁺] 626.3489, found626.3489.

Example 18

Migrastatin 1: To a solution of TBS-migrastatin 37 (19 mg, 0.032 mmol)in THF (1.5 mL) at rt was added HF•pyridine (0.25 mL). After stirringfor 15 h, the reaction mixture was carefully treated with MeOTMS (3 mL)and concentrated under reduced pressure. Purification of the crudeproduct by FC (hexane/EtOAc 2:1→1:1→1:2) afforded migrastatin 1 (13 mg,85%) as a white solid. [α]_(D) +12.6° (c 0.50, MeOH); ¹H-NMR (500 MHz,CDCl₃) δ 7.82 (br s, 1H), 6.49 (ddd, J=15.7, 10.5, 3.7, 1H), 5.64 (dd,J=10.7, 1.2, 1H), 5.58 (dd, J=15.7, 1.2, 1H), 5.54-5.48 (m, 1H), 5.24(dd, J=15.5, 4.7, 1H), 5.08 (d, J=10.0, 1H), 3.47 (dd, J=8.7, 4.7, 1H),3.30 (s, 3H), 3.03 (dd, J=8.7, 1.7, 1H), 2.99-2.87 (m, 2H), 2.80 (br s,1H), 2.73-2.68 (m, 2H), 2.50 (app t, J=6.9, 2H), 2.44-2.39 (m, 2H),2.28-2.17 (m, 4H), 2.16-2.08 (m, 1H), 1.86 (d, J=1.2, 3H), 1.69-1.55 (m,2H), 1.41-1.30 (m, 2H), 1.12 (d, J=7.2, 3H), 0.96 (d, J=6.9, 3H);¹³C-NMR (125 MHz, CDCl₃) δ 210.88, 171.78, 163.86, 150.01, 132.99,131.17, 130.46, 127.87, 122.08, 82.39, 77.92, 76.92, 56.93, 51.18,39.88, 37.68, 37.66, 34.12, 31.93, 31.08, 30.34, 30.09, 25.99, 20.09,13.39; MS (ESI) 512 [M+Na⁺]; HRMS (FAB) calcd. for C₂₇H₃₉NO₇Na [M+Na⁺]512.2624, found 512.2604.

Example 19

6-Heptenoyl Chloride 38: To a solution of 6-heptenoic acid (251 μL, 1.85mmol) in CH₂Cl₂ (5 mL) at rt was added oxalyl chloride (476 μL, 5.55mmol) and DMF (1 drop). After stirring for 1 hr, the reaction mixturewas concentrated under reduced pressure and put under high vacuum for 15min. The residual yellow oil was dissolved in CH₂Cl₂ (3 mL) and used asa stock solution (0.62M) for the subsequent acylation reactions.

Example 20

Ester 39: To a solution of alcohol 34 (37 mg, 0.070 mmol) in CH₂Cl₂ (2mL) at rt was added DMAP (17 mg, 0.139 mmol) and 6-heptenoyl chloride(See above for the preparation of a stock solution of 6-heptenoylchloride 38 in CH₂Cl₂) 38 (202 μL, 0.125 mmol, 0.62M in CH₂Cl₂). Afterstirring for 2 h, the reaction mixture was treated with 0.1M HCl anddiluted with CH₂Cl₂. The organic layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (3×). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure. Purification ofthe crude product by FC (hexane/EtOAc 10:1→4:1→2:1) afforded ester 39(31 mg, 69%) as a colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 8.22 (br s,1H), 5.79-5.72 (m, 1H), 5.67-5.61 (m, 2H), 5.55 (d, J=10.3, 1H), 5.36(dd, J=10.3, 1.3, 1H), 5.30 (d, J=17.1, 1H), 5.00-4.92 (m, 2H),3.40-3.37 (m, 2H), 3.20 (s, 3H), 2.93-2.85 (m, 2H), 2.71 (dd, J=17.0,4.0, 2H), 2.55-2.42 (m, 2H), 2.29-2.21 (m, 2H), 2.19-2.11 (m, 3H),2.09-2.00 (m, 2H), 1.60 (s, 3H), 1.59-1.51 (m, 5H), 1.47-1.42 (m, 1H),1.38-1.32 (m, 2H), 0.92-0.90 (m, 15H), 0.05 (s, 3H), −0.01 (s, 3H);¹³C-NMR (125 MHz, CDCl₃) δ 211.06, 172.07, 171.68, 138.28, 137.75,134.48, 126.86, 119.24, 114.73, 86.25, 78.39, 73.62, 56.01, 47.13,41.67, 37.70, 34.18, 34.08, 33.78, 33.28, 30.41, 28.21, 28.18, 26.11,24.38, 20.12, 18.56, 17.61, 13.73, 12.64, −3.78, −4.97; MS (ESI) 656[M+Na⁺]; HRMS (FAB) calcd. for C₃₅H₅₉NO₇SiNa [M+Na⁺] 656.3959, found656.3956.

Example 21

TBS-2,3-Dihydromigrastatin 40: To a solution of ester 39 (31 mg, 0.048mmol) in refluxing toluene (100 mL) was added Grubbs-II catalyst 16 (8mg, 0.0094 mmol). After stirring for 15 min, the reaction mixture wascooled to rt and filtered through a silica plug (hexane/EtOAc 1:3).Purification of the crude product by FC (hexane/EtOAc 5:1→2:1→1:1)afforded TBS-2,3-dihydromigrastatin 40 (23 mg, 79%) as a colorless oil.¹H-NMR (500 MHz, CDCl₃) δ 7.90 (br s, 1H), 5.65-5.57 (m, 2H), 5.35 (dd,J=15.7, 5.1, 1H), 5.20 (d, J=9.2, 1H), 3.43-3.40 (m, 1H), 3.23-3.20 (m,1H), 3.21 (s, 3H), 3.03-2.98 (m, 1H), 2.95-2.91 (m, 1H), 2.73-2.69 (m,2H), 2.59-2.43 (m, 2H), 2.33-2.22 (m, 4H), 2.17-2.07 (m, 3H), 1.75 (d,J=0.9, 3H), 1.61-1.55 (m, 5H), 1.40-1.35 (m, 3H), 1.07 (d, J=7.2, 3H),0.94 (d, J=6.8, 3H), 0.91 (s, 9H), 0.07 (s, 3H), 0.03 (s, 3H); ¹³C-NMR(125 MHz, CDCl₃) δ 210.63, 171.84, 171.80, 134.70, 131.22, 129.51,128.39, 82.98, 79.10, 76.46, 56.49, 51.24, 40.62, 37.74, 37.69, 34.18,33.35, 33.18, 31.11, 30.37, 26.29, 25.76, 25.16, 22.80, 20.18, 18.71,13.66, 13.12, −3.56, −4.97; MS (ESI) 628 [M+Na⁺]; HRMS (FAB) calcd. forC₃₃H₅₅NO₇Na [M+Na⁺] 628.3646, found 628.3644.

Example 22

2,3-Dihydromigrastatin 41: To a solution of TBS-2,3-dihydromigrastatin40 (23 mg, 0.038 mmol) in THF (1.5 mL) at rt was added HF•pyridine (0.3mL). After stirring for 15 h, the reaction mixture was carefully treatedwith MeOTMS (4 mL) and concentrated under reduced pressure. Purificationof the crude product by FC (hexane/EtOAc 2:1→1:1→1:2) afforded2,3-dihydromigrastatin 41 (15 mg, 81%) as a white foam. ¹H-NMR (500 MHz,CDCl₃) δ 7.97 (br s, 1H), 5.68-5.60 (m, 2H), 5.34 (dd, J=15.6, 5.6, 1H),5.19 (d, J=9.7, 1H), 3.49-3.46 (m, 1H), 3.33 (s, 3H), 3.22 (app d,J=9.1, 1H), 3.07-3.00 (m, 1H), 2.98-2.91 (m, 1H), 2.72 (dd, J=17.1, 2.3,2H), 2.59-2.50 (m, 1H), 2.49-2.40 (m, 1H), 2.30-2.04 (m, 7H), 1.79 (d,J=1.3, 3H), 1.63-1.56 (m, 4H), 1.55-1.48 (m, 1H), 1.42-1.35 (m, 3H),1.09 (d, J=7.2, 3H), 0.99 (d, J=6.9, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ210.64, 171.88, 171.73, 133.95, 132.35, 130.27, 128.05, 81.92, 77.42,76.45, 56.70, 51.38, 40.37, 37.73, 37.68, 34.15, 32.50, 31.72, 30.45,30.35, 25.94, 24.80, 22.33, 20.16, 13.22, 13.20; MS (ESI) 514 [M+Na⁺];HRMS (FAB) calcd. for C₂₇H₄₁NO₇Na [M+Na⁺] 514.2781, found 514.2768.

Example 23

N-Methyl-2,3-Dihydromigrastatin 42: To a solution of2,3-dihydromigrastatin 41 (4 mg, 0.0081 mmol) in acetone (0.4 mL) at rtwas added MeI (excess) and Cs₂CO₃ (excess). After stirring for 4 h, thereaction mixture was concentrated under reduced pressure to a volume ofca. 0.2 mL. Purification of the residual solution by preparative TLC(hexane/EtOAc 1:2) afforded N-methyl-2,3-dihydromigrastatin 42 (3.5 mg,85%) as a colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 5.68-5.61 (m, 2H),5.34 (dd, J=15.6, 5.6, 1H), 5.19 (d, J=9.6, 1H), 3.49-3.46 (m, 1H), 3.33(s, 3H), 3.22 (app d, J=9.1, 1H), 3.14 (s, 3H), 3.07-3.01 (m, 1H),2.95-2.89 (m, 1H), 2.82-2.78 (m, 2H), 2.58-2.50 (m, 1H), 2.49-2.42 (m,1H), 2.33-2.27 (m, 2H), 2.25-2.04 (m, 5H), 1.79 (d, J=1.3, 3H),1.65-1.52 (m, 5H), 1.47-1.42 (m, 1H), 1.37-1.31 (m, 2H), 1.09 (d, J=7.2,3H), 0.99 (d, J=6.9, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 210.67, 172.20,171.72, 133.93, 132.35, 130.33, 128.06, 81.92, 77.45, 76.47, 56.72,51.38, 40.44, 38.75, 38.71, 34.27, 32.51, 31.73, 30.47, 29.34, 26.36,25.94, 24.81, 22.33, 20.09, 13.23; MS (ESI) 528 [M+Na⁺]; HRMS (FAB)calcd. for C₂₈H₄₃NO₇Na [M+Na⁺] 528.2937, found 528.2939.

Example 24

Unsaturated Ester 43: To a solution of alcohol 26 (109 mg, 0.346 mmol)in toluene (1 mL) at rt was added pyridine (84 μL, 1.04 mmol) and themixed anhydride (See above for the preparation of a stock solution ofthe mixed anhydride in toluene) (1 mL, 0.535 mmol, 0.54M in toluene).After stirring for 12 h, the reaction mixture was filtered through asilica plug (hexane/EtOAc 30:1). Purification of the crude product by FC(pentane/CH₂Cl₂ 3:1→2:1) afforded unsaturated ester 43 (70 mg, 48%) as acolorless oil. [α]_(D) +2.6° (c 1.00, CHCl₃); IR (CHCl₃) 2934, 2882,2851, 1705, 1653, 1470, 1381, 1246, 1126, 1079, 1026; ¹H-NMR (500 MHz,CDCl₃) δ 6.99-6.93 (m, 1H), 5.86-5.76 (m, 2H), 5.66-5.59 (m, 1H), 5.44(d, J=9.5, 1H), 5.29-5.22 (m, 2H), 5.07-4.99 (m, 2H), 4.61 (d, J=12.1,1H), 4.57 (d, J=12.1, 1H), 3.47 (dd, J=7.2, 2.9, 1H), 3.37 (app t,J=7.7, 1H), 3.19 (s, 3H), 2.63-2.59 (m, 1H), 2.33-2.28 (m, 2H),2.24-2.19 (m, 2H), 1.73 (d, J=1.3, 3H), 0.91 (d, J=6.6, 3H), 0.90 (s,9H), 0.05 (s, 3H), 0.02 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 166.57,148.47, 137.05, 135.58, 135.10, 128.21, 121.52, 118.79, 115.53, 86.26,78.37, 63.07, 56.06, 34.26, 32.02, 31.48, 26.15, 21.49, 18.54, 13.96,−3.80, −4.85; MS (ESI) 445 [M+Na⁺]; HRMS (FAB) calcd. for C₂₄H₄₃O₄Si[M+H⁺] 423.2931, found 423.2929.

Example 25

TBS-Migrastatin Core 44: To a solution of unsaturated ester 43 (35 mg,0.083 mmol) in refluxing toluene (125 mL) was added Grubbs-II catalyst16 (14 mg, 0.017 mmol). After stirring for 15 min, the reaction mixturewas cooled to rt and filtered through a silica plug (hexane/EtOAc 4:1).Purification of the crude product by FC (hexane/EtOAc 20:1) affordedTBS-migrastatin core 44 (18 mg, 55%) as a colorless oil. ¹H-NMR (500MHz, CDCl₃) δ 6.85-6.79 (m, 1H), 5.74 (d, J=15.9, 1H), 5.56-5.50 (m,2H), 5.12 (dd, J=15.5, 8.7, 1H), 4.68 (d, J=15.8, 1H), 4.62 (d, J=15.8,1H), 3.44 (dd, J=8.3, 1.4, 1H), 3.33-3.30 (m, 1H), 3.17 (s, 3H),3.03-2.97 (m, 1H), 2.47-2.36 (m, 2H), 2.31-2.24 (m, 1H), 2.21-2.14 (m,1H), 1.64 (s, 3H), 0.92 (s, 9H), 0.83 (d, J=6.8, 3H), 0.07 (s, 3H), 0.06(s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 165.37, 149.91, 131.98, 130.48,126.58, 121.83, 117.57, 85.82, 77.49, 65.56, 55.83, 33.11, 32.46, 30.01,26.27, 22.17, 18.71, 12.90, −3.57, −5.02; MS (ESI) 417 [M+Na⁺]; HRMS(FAB) calcd. for C₂₂H₃₈O₄SiNa [M+Na⁺] 417.2437, found 417.2456.

Example 26

Migrastatin Core 45: To a solution of TBS-migrastatin core 44 (18 mg,0.0457 mmol) in THF (1.5 mL) at rt was added HF•pyridine (0.3 mL). Afterstirring for 14 h, the reaction mixture was carefully treated withMeOTMS (4 mL) and concentrated under reduced pressure. Purification ofthe crude product by FC (hexane/EtOAc 10:1→5:1) afforded migrastatincore 45 (8.5 mg, 66%) as a colorless oil. [α]_(D) +106.0° (c 0.50,CHCl₃); IR (CHCl₃) 3567, 2933, 2881, 1716, 1602, 1448, 1393, 1255, 1107,1052; ¹H-NMR (500 MHz, CDCl₃) δ 6.81-6.75 (m, 1H), 5.73 (d, J=15.9, 1H),5.62-5.55 (m, 2H), 5.14 (dd, J=15.2, 6.8, 1H), 4.72 (d, J=15.6, 1H),4.63 (d, J=15.6, 1H), 3.42-3.38 (m, 2H), 3.28 (s, 3H), 3.03-2.97 (m,1H), 2.69 (br s, 1H), 2.47-2.38 (m, 2H), 2.32-2.18 (m, 2H), 1.68 (s,3H), 0.88 (d, J=6.9, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 165.36, 149.52,133.85, 129.79, 129.51, 127.50, 122.15, 84.62, 76.09, 65.40, 56.25,32.20, 31.34, 29.99, 22.27, 12.66; MS (ESI) 303 [M+Na⁺]; HRMS (FAB)calcd. for C₁₆H₂₄O₄Na [M+Na⁺] 303.1571, found 303.1572.

Example 27

Ester 46: To a solution of alcohol 26 (275 mg, 0.874 mmol) in CH₂Cl₂ (3mL) at rt was added DMAP (214 mg, 1.75 mmol) and 6-heptenoyl chloride(See above for the preparation of a stock solution of 6-heptenoylchloride 38 in CH₂Cl₂) 38 (2.5 mL, 1.57 mmol, 0.62M in CH₂Cl₂). Afterstirring for 20 min, the reaction mixture was treated with 0.1M HCl anddiluted with CH₂Cl₂. The organic layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (3×). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure. Purification ofthe crude product by FC (hexane/EtOAc 30:1) afforded ester 46 (302 mg,82%) as a colorless oil. [α]_(D) +3.0° (c 0.50, CHCl₃); IR (CHCl₃) 2980,2933, 2863, 1722, 1458, 1382, 1252, 1112, 1024; ¹H-NMR (500 MHz, CDCl₃)δ 5.83-5.75 (m, 1H), 5.66-5.59 (m, 1H), 5.43 (d, J=9.5, 1H), 5.30-5.23(m, 2H), 5.03-4.94 (m, 2H), 4.56 (d, J=12.0, 1H), 4.51 (d, J=12.0, 1H),3.46 (dd, J=7.2, 2.9, 1H), 3.37 (app t, J=7.7, 1H), 3.20 (s, 3H),2.61-2.57 (m, 1H), 2.32 (app t, J=7.5, 2H), 2.06 (app q, J=7.2, 2H),1.74 (d, J=1.2, 3H), 1.68-1.62 (m, 2H), 1.45-1.39 (m, 2H), 0.91 (s, 9H),0.90 (d, J=6.5, 3H), 0.05 (s, 3H), 0.02 (s, 3H); ¹³C-NMR (125 MHz,CDCl₃) δ 173.65, 138.40, 135.59, 135.10, 128.12, 118.77, 114.67, 86.24,78.36, 63.11, 56.07, 34.24, 34.17, 33.35, 28.35, 26.15, 24.46, 21.45,18.54, 13.98, −3.80, −4.85; MS (ESI) 447 [M+Na⁺]; HRMS (FAB) calcd. forC₂₄H₄₄O₄SiNa [M+Na⁺] 447.2906, found 447.2893.

Example 28

TBS-Macrolactone 47: To a solution of ester 46 (95 mg, 0.224 mmol) inrefluxing toluene (450 mL) was added Grubbs-II catalyst 16 (38 mg, 0.045mmol). After stirring for 15 min, the reaction mixture was cooled to rtand filtered through a silica plug (hexane/EtOAc 5:1). Purification ofthe crude product by FC (hexane/EtOAc 30:1) afforded TBS-macrolactone 47(67 mg, 76%) as a colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 5.71-5.65 (m,1H), 5.56 (d, J=10.0, 1H), 5.28 (dd, J=15.7, 8.0, 1H), 4.52 (d, J=13.9,1H), 4.35 (d, J=13.9, 1H), 3.46 (dd, J=7.7, 2.6, 1H), 3.39 (app t,J=7.8, 1H), 3.20 (s, 3H), 2.85-2.82 (m, 1H), 2.42-2.36 (m, 1H),2.26-2.20 (m, 1H), 2.18-2.14 (m, 1H), 2.11-2.06 (m, 1H), 1.77-1.72 (m,1H), 1.71 (d, J=1.1, 3H), 1.62-1.50 (m, 2H), 1.46-1.40 (m, 1H), 0.91 (s,9H), 0.88 (d, J=6.8, 3H), 0.07 (s, 3H), 0.05 (s, 3H); ¹³C-NMR (125 MHz,CDCl₃) δ 173.74, 134.81, 133.62, 128.75, 126.14, 85.42, 77.78, 65.01,55.97, 34.31, 34.01, 29.37, 27.34, 26.16, 23.36, 23.09, 18.58, 13.86,−3.78, −4.96; MS (ESI) 419 [M+Na⁺]; HRMS (FAB) calcd. for C₂₂H₄OO₄SiNa[M+Na⁺] 419.2594, found 419.2601.

Example 29

Macrolactone 48: To a solution of TBS-macrolactone 47 (179 mg, 0.452mmol) in THF (6 mL) at rt was added HF•pyridine (in the beginning: 0.6mL, after a total of 15 h: an additional 0.6 mL, after a total of 25 h:an additional 0.3 mL). After stirring for a total of 40 h, the reactionmixture was carefully treated with MeOTMS (12 mL) and concentrated underreduced pressure. Purification of the crude product by FC (hexane/EtOAc10:1→5:1) afforded macrolactone 48 (120 mg, 94%) as a white crystallinesolid. [α]_(D) +115.3° (c 1.00, CHCl₃); IR (CHCl₃) 3567, 3016, 2933,2858, 1724, 1450, 1387, 1317, 1258, 1145, 1115, 979; ¹H-NMR (500 MHz,CDCl₃) δ 5.74-5.67 (m, 2H), 5.23 (dd, J=15.7, 7.7, 1H), 4.54 (d, J=13.1,1H), 4.29 (d, J=13.1, 1H), 3.46-3.39 (m, 2H), 3.30 (s, 3H), 2.82-2.77(m, 1H), 2.44-2.39 (m, 1H), 2.26-2.15 (m, 2H), 2.03-1.97 (m, 1H), 1.74(d, J=0.9, 3H), 1.74-1.70 (m, 1H), 1.60-1.52 (m, 2H), 1.36-1.32 (m, 1H),0.93 (d, J=6.9, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 173.69, 135.19, 134.39,129.02, 127.14, 83.82, 75.91, 64.76, 56.34, 34.23, 32.06, 29.88, 27.20,23.40, 23.27, 12.81; MS (ESI) 305 [M+Na⁺]; HRMS (FAB) calcd. forC₁₆H₂₆O₄Na [M+Na⁺] 305.1719, found 305.1729.

Example 30

Acetylated Macrolactone 49: To a solution of macrolactone 48 (4.5 mg,0.016 mmol) in CH₂Cl₂ (0.75 mL) at rt was added DMAP (6 mg, 0.048 mmol)and AcCl (3.5 μL, 0.048 mmol). After stirring for 24 h, the reactionmixture was concentrated under reduced pressure to a volume of ca. 0.2mL. Purification of the residual solution by preparative TLC(hexane/EtOAc 2:1) afforded the acetylated macrolactone 49 (4 mg, 76%)as a colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 5.78-5.72 (m, 1H), 5.37(dd, J=15.7, 8.2, 1H), 5.28 (d, J=10.0, 1H), 4.89 (dd, J=8.0, 3.6, 1H),4.56 (d, J=13.2, 1H), 4.32 (d, J=13.2, 1H), 3.57 (app t, J=8.1, 1H),3.23 (s, 3H), 3.02-2.97 (m, 1H), 2.46-2.41 (m, 1H), 2.25-2.19 (m, 2H),2.11 (s, 3H), 2.10-2.05 (m, 1H), 1.81-1.75 (m, 1H), 1.71 (d, J=0.9, 3H),1.61-1.53 (m, 2H), 1.43-1.39 (m, 1H), 0.95 (d, J=6.9, 3H); ¹³C-NMR (125MHz, CDCl₃) δ 173.61, 170.82, 135.23, 132.14, 127.76, 82.63, 76.83,64.69, 56.46, 34.30, 32.10, 29.58, 27.02, 23.39, 23.04, 21.10, 14.85; MS(ESI) 347 [M+Na⁺]; HRMS (FAB) calcd. for C₁₈H₂₈O₅Na [M+Na⁺] 347.1834,found 347.1848.

Example 31

Oxidized Macrolactone 50: To a solution of macrolactone 48 (7 mg, 0.025mmol) in CH₂Cl₂ (1.5 mL) at rt was added Dess-Martin periodinane (12 mg,0.027 mmol). After stirring for 4 h, the reaction mixture wasconcentrated under reduced pressure to a volume of ca. 0.2 mL.Purification of the residual solution by preparative TLC (hexane/EtOAc1:1) afforded oxidized macrolactone 50 (5 mg, 72%) as a colorless oil.¹H-NMR (500 MHz, CDCl₃) δ 5.92-5.86 (m, 1H), 5.73 (d, J=9.9, 1H), 5.34(dd, J=15.5, 8.0, 1H), 4.54 (d, J=11.6, 1H), 4.37 (d, J=8.0, 1H), 4.31(d, J=11.6, 1H), 3.71-3.65 (m, 1H), 3.32 (s, 3H), 2.41-2.36 (m, 1H),2.27-2.21 (m, 1H), 2.20-2.16 (m, 1H), 2.06-1.99 (m, 1H), 1.81 (s, 3H),1.68-1.60 (m, 2H), 1.58-1.51 (m, 1H), 1.41-1.33 (m, 1H), 1.19 (d, J=7.1,3H); ¹³C-NMR (125 MHz, CDCl₃) δ 207.91, 173.74, 138.30, 130.64, 130.42,124.64, 86.15, 62.75, 56.65, 41.61, 34.04, 30.35, 26.64, 23.40, 23.18,18.89; MS (ESI) 303 [M+Na⁺]; HRMS (FAB) calcd. for C₁₆H₂₄O₄Na [M+Na⁺]303.1572, found 303.1588.

Example 32

Hydrolyzed Core 51: To a solution of macrolactone 48 (5 mg, 0.018 mmol)in MeOH (1.5 mL) at rt was added 0.5M NaOH (0.3 mL). After stirring for2 h, the reaction mixture was concentrated under reduced pressure to avolume of ca. 0.5 mL, diluted with CH₂Cl₂, and acidified with 1M HCl (2mL). The organic layer was separated, dried (MgSO₄), and concentratedunder reduced pressure to afford hydrolyzed core 51 (4 mg, 77%) as acolorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 5.75-5.70 (m, 1H), 5.34 (dd,J=15.5, 8.7, 1H), 5.23 (d, J=10.1, 1H), 4.15 (d, J=11.8, 1H), 3.97 (d,J=11.8, 1H), 3.47-3.44 (m, 1H), 3.29-3.26 (m, 1H), 3.23 (s, 3H),2.74-2.69 (m, 1H), 2.36 (app t, J=7.4, 2H), 2.14 (app q, J=7.1, 2H),1.82 (d, J=1.2, 3H), 1.69-1.63 (m, 2H), 1.51-1.45 (m, 2H), 0.99 (d,J=6.7, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 177.87, 136.20, 134.96, 130.90,127.34, 83.24, 77.55, 61.56, 55.65, 34.56, 33.59, 31.81, 28.38, 24.13,22.00, 16.39; MS (ESI) 323 [M+Na⁺]; HRMS (FAB) calcd. for C₁₆H₂₈O₅Na[M+Na⁺] 323.1834, found 323.1840.

Example 33

Allylic Azide 52: To a solution of alcohol 26 (300 mg, 0.954 mmol) intoluene (3 mL) at rt was added DBU (214 μL, 1.43 mmol) anddiphenylphosphoryl azide (308 μL, 1.43 mmol). After stirring for 5 h,the reaction mixture was treated with saturated aqueous NH₄Cl solutionand diluted with Et₂O. The organic layer was separated and the aqueouslayer was extracted with Et₂O (3×). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure. Purification ofthe crude product by FC (hexane/EtOAc 30:1) afforded allylic azide 52(281 mg, 87%) as a colorless oil. Compound 52 should be used immediatelyfor the subsequent steps to avoid double bond isomerization. ¹H-NMR (500MHz, CDCl₃) δ 5.68-5.60 (m, 1H), 5.52 (d, J=10.0, 1H), 5.32-5.25 (m,2H), 3.81 (d, J=13.0, 1H), 3.66 (d, J=13.0, 1H), 3.45 (dd, J=7.1, 3.0,1H), 3.39 (app t, J=7.5, 1H), 3.21 (s, 3H), 2.56-2.52 (m, 1H), 1.77 (d,J=1.2, 3H), 0.93-0.90 (m, 12H), 0.06 (s, 3H0, 0.04 (s, 3H); ¹³C-NMR (125MHz, CDCl₃) δ 135.97, 135.19, 127.27, 118.81, 86.04, 78.39, 56.13,51.46, 34.40, 26.14, 22.21, 18.53, 14.43, −3.80, −4.77; MS (ESI) 362[M+Na⁺]; HRMS (FAB) calcd. for C₁₇H₃₃N₃O₂SiNa [M+Na⁺] 362.2240, found362.2239.

Example 34

Amide 53: To a solution of azide 52 (184 mg, 0.542 mmol) in THF (5 mL)at 70° C. was added PPh₃ (249 mg, 0.949 mmol) and H₂O (49 μL, 2.71mmol). After stirring for 4 h, the reaction mixture was dried (MgSO₄)and concentrated under reduced pressure. The residue was dissolved inCH₂Cl₂ (5 mL) and treated with i-Pr₂NEt (378 μL, 2.17 mmol), 6-heptenoicacid (147 μL, 1.08 mmol), and EDC (207 mg, 1.08 mmol). After stirringfor 30 min, the reaction mixture was concentrated under reduced pressureto a volume of ca. 1 mL. Purification of the residual solution byFC(CH₂Cl₂→CH₂Cl₂/Et₂O 10:1) afforded amide 53 (211 mg, 92%) as acolorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 5.83-5.74 (m, 1H), 5.70-5.64(m, 1H), 5.41 (br s, 1H), 5.32-5.23 (m, 3H), 5.01-4.93 (m, 2H), 3.86(dd, J=14.1, 5.6, 1H), 3.79 (dd, J=14.1, 5.5, 1H), 3.47-3.37 (m, 2H),3.21 (s, 3H), 2.61-2.56 (m, 1H), 2.19-2.15 (m, 2H), 2.08-2.04 (m, 2H),1.68 (d, J=1.3, 3H), 1.67-1.59 (m, 2H), 1.45-1.38 (m, 2H), 0.91-0.89 (m,12H), 0.06 (s, 3H), 0.02 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 172.79,138.44, 135.10, 133.94, 129.82, 118.66, 114.66, 86.01, 78.22, 56.11,39.84, 36.65, 34.26, 33.44, 28.52, 26.11, 25.25, 21.93, 18.50, 14.76,−3.83, −4.77; MS (ESI) 446 [M+Na⁺]; HRMS (FAB) calcd. for C₂₄H₄₅NO₃SiNa[M+Na⁺] 446.3066, found 446.3065.

Example 35

TBS-Macrolactam 54: To a solution of amide 53 (105 mg, 0.248 mmol) inrefluxing toluene (350 mL) was added Grubbs-II catalyst 16 (42 mg, 0.050mmol). After stirring for 15 min, the reaction mixture was cooled to rtand filtered through a silica plug (hexane/EtOAc 1:2). Purification ofthe crude product by FC (hexane/EtOAc 2:1) afforded TBS-macrolactam 54(59 mg, 60%) as a colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 5.81-5.75 (m,1H), 5.46 (d, J=9.9, 1H), 5.36 (dd, J=15.9, 6.0, 1H), 5.30 (br s, 1H),3.77 (dd, J=13.9, 3.5, 1H), 3.66 (dd, J=13.9, 5.4, 1H), 3.48-3.44 (m,2H), 3.21 (s, 3H), 2.63-2.58 (m, 1H), 2.21-2.08 (m, 3H), 2.05-1.98 (m,1H), 1.73 (d, J=1.1, 3H), 1.65-1.49 (m, 3H), 1.39-1.32 (m, 1H),0.92-0.90 (m, 12H), 0.07 (s, 3H), 0.05 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃)δ 173.26, 134.11, 133.90, 129.03, 128.54, 84.80, 77.46, 56.29, 41.41,36.01, 34.48, 29.59, 27.45, 26.11, 24.68, 24.32, 18.56, 14.77, −3.92,−4.93; MS (ESI) 418 [M+Na⁺]; HRMS (FAB) calcd. for C₂₂H₄₁NO₃SiNa [M+Na⁺]418.2753, found 418.2752.

Example 36

Macrolactam 55: To a solution of TBS-macrolactam 54 (91 mg, 0.230 mmol)in THF (3 mL) at rt was added HF•pyridine (in the beginning: 0.4 mL,after a total of 18 h: an additional 0.15 mL). After stirring for atotal of 21 h, the reaction mixture was carefully treated with MeOTMS (5mL) and concentrated under reduced pressure. Purification of the crudeproduct by FC (hexane/EtOAc 1:1→1:2) afforded macrolactam 55 (52 mg,81%) as a colorless oil. [α]_(D) +101.3° (c 1.00, CHCl₃); IR (CHCl₃)3566, 3444, 3021, 2936, 2828, 1658, 1504, 1478, 1398, 1229, 1088, 979;¹H-NMR (500 MHz, CDCl₃) δ 5.79-5.73 (m, 1H), 5.66 (d, J=10.2, 1H), 5.24(dd, J=15.8, 7.5, 1H), 5.12 (br s, 1H), 3.91 (dd, J=13.7, 4.1, 1H),3.50-3.46 (m, 2H), 3.34-3.30 (m, 1H), 3.31 (s, 3H), 2.89 (br s, 1H),2.56-2.52 (m, 1H), 2.32-2.25 (m, 2H), 2.16-2.11 (m, 1H), 1.96-1.89 (m,1H), 1.77 (d, J=1.1, 3H), 1.73-1.51 (m, 3H), 1.37-1.32 (m, 1H), 0.94 (d,J=6.9, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 173.36, 135.52, 133.77, 129.89,128.73, 83.21, 76.38, 56.45, 41.40, 35.95, 32.27, 29.86, 27.00, 24.82,24.42, 13.03; MS (ESI) 304 [M+Na⁺]; HRMS (FAB) calcd. for C₁₆H₂₇NO₃Na[M+Na⁺] 304.1888, found 304.1889.

Example 37

Allylic Bromide 56: To a solution of alcohol 26 (325 mg, 1.03 mmol) inCH₂Cl₂ (10 mL) at rt was added solid supported PPh₃ (excess untilreaction complete) and CBr₄ (478 mg, 1.44 mmol). After stirring for 15min, the reaction mixture was filtered through a cotton plug andconcentrated under reduced pressure to yield the allylic bromide 56.¹H-NMR (500 MHz, CDCl₃) δ 5.67 (ddd, J=17.2, 10.3, 8.3, 1H), 5.41 (dd,J=10.0, 0.9, 1H), 5.31 (dd, J=10.3, 2.0, 1H), 5.27 (dt, J=17.2, 1.0,1H), 3.94 (s, 2H), 3.55 (dd, J=7.2, 3.0, 1H), 3.39 (app t, J=7.4, 1H),3.21 (s, 3H), 2.63-2.56 (m, 1H), 1.81 (d, J=0.9, 3H), 0.93 (d, J=6.4,3H), 0.91 (s, 9H), 0.06 (s, 3H), 0.03 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃)δ 136.35, 135.20, 129.64, 118.82, 86.09, 77.57, 56.15, 34.68, 32.34,26.13, 21.91, 18.50, 13.54, −3.83, −4.82.

Example 38

β-Ketosulfone 57: To a solution of methyl phenyl sulfone (1.43 g, 9.14mmol) in THF (15 mL) at −15° C. was added BuLi (6.28 mL, 10.0 mmol, 1.6Min hexane). After stirring for 30 min, the reaction mixture was cooledto −78° C. and ethyl 6-heptenoate (802 μL, 4.57 mmol) was added. Thereaction mixture was warmed to rt and then treated with saturatedaqueous NH₄Cl solution. The organic layer was separated and the aqueouslayer was extracted with EtOAc (3×). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure. Purification ofthe crude product by FC (hexane/EtOAc 5:1) afforded β-ketosulfone 57(1.12 g, 92%) as a white solid. ¹H-NMR (500 MHz, CDCl₃) δ 7.88-7.86 (m,2H), 7.68-7.65 (m, 1H), 7.58-7.54 (m, 2H), 5.79-5.71 (m, 1H), 5.00-4.92(m, 2H), 4.14 (s, 2H), 2.70-2.67 (m, 2H), 2.05-2.00 (m, 2H), 1.58-1.52(m, 2H), 1.38-1.32 (m, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ 197.99, 138.14,134.19, 129.25, 129.06, 128.17, 114.73, 66.70, 44.12, 33.26, 27.85,22.42; MS (ESI) 289 [M+Na⁺]; HRMS (FAB) calcd. for C₁₄H₁₈O₃SNa [M+Na⁺]289.0874, found 289.0882.

Example 39

Ketone 58: To a solution of β-ketosulfone 57 (685 mg, 2.57 mmol) intoluene (5 mL) at rt was added DBU (385 μL, 2.57 mmol). After stirringfor 50 min, a solution of crude allylic bromide 56 in toluene (5 mL) wasadded and the reaction mixture was stirred for another 45 min. Thereaction mixture was concentrated under reduced pressure to a volume ofca. 1 mL and the residual solution was filtered through a silica plug(hexane/EtOAc 7:1). To a solution of crude alkylated sulfone in MeOH (10mL) at rt was added Na₂HPO₄ (366 mg, 2.57 mmol) and 10% Na/Hg (474 mg,ca. 2.06 mmol). After stirring for 3 h, the reaction mixture wasfiltered through a cotton plug and H₂O was added to the filtrate. Theorganic layer was separated and the aqueous layer was extracted withCH₂Cl₂ (3×). The combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure. Purification of the crude productby FC (hexane/EtOAc 30:1) afforded ketone 58 (258 mg, 61%) as acolorless oil. ¹H-NMR (500 MHz, CDCl₃) δ 5.82-5.75 (m, 1H), 5.67-5.60(m, 1H), 5.29-5.18 (m, 3H), 5.02-4.93 (m, 2H), 3.41 (dd, J=7.2, 2.8,1H), 3.37 (app t, J=7.6, 1H), 3.20 (s, 3H), 2.52-2.47 (m, 1H), 2.44-2.38(m, 4H), 2.28-2.18 (m, 2H), 2.06 (app q, J=7.1, 2H), 1.64 (d, J=1.2,3H), 1.62-1.56 (m, 2H), 1.41-1.35 (m, 2H), 0.90 (s, 9H), 0.87 (d, J=6.7,3H), 0.05 (s, 3H), 0.01 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 210.57,138.45, 135.30, 131.81, 131.30, 118.53, 114.64, 86.27, 78.61, 56.10,42.64, 41.23, 34.05, 33.50, 28.46, 26.16, 26.12, 23.27, 23.11, 18.55,14.05, −3.79, −4.79; MS (ESI) 445 [M+Na⁺]; HRMS (FAB) calcd. forC₂₅H₄₆O₃SiNa [M+Na⁺] 445.3114, found 445.3095.

Example 40

TBS-Macroketone 59: To a solution of ketone 58 (258 mg, 0.610 mmol) inrefluxing toluene (1200 mL) was added Grubbs-II catalyst 16 (104 mg,0.122 mmol). After stirring for 15 min, the reaction mixture was cooledto rt and filtered through a silica plug (hexane/EtOAc 2:1).Purification of the crude product by FC (hexane/EtOAc 20:1) affordedTBS-macroketone 59 (194 mg, 81%) as a colorless oil. ¹H-NMR (500 MHz,CDCl₃) δ 5.67-5.61 (m, 1H), 5.32 (dd, J=15.7, 6.7, 1H), 5.26 (dd, J=9.8,0.9, 1H), 3.41-3.36 (m, 2H), 3.21 (s, 3H), 2.55-2.49 (m, 1H), 2.46-2.41(m, 1H), 2.39-2.33 (m, 1H), 2.32-2.18 (m, 5H), 2.14-2.10 (m, 1H),1.68-1.63 (m, 1H), 1.67 (d, J=1.3, 3H), 1.62-1.53 (m, 2H), 1.51-1.46 (m,1H), 0.90 (s, 9H), 0.89 (d, J=6.8, 3H), 0.05 (s, 3H), 0.00 (s, 3H);¹³C-NMR (125 MHz, CDCl₃) δ 211.96, 133.10, 131.91, 131.68, 129.87,84.77, 79.32, 56.24, 41.44, 40.91, 34.32, 30.25, 28.74, 26.84, 26.15,23.15, 22.85, 18.60, 12.78, −3.85, −5.03; MS (ESI) 417 [M+Na⁺]; HRMS(FAB) calcd. for C₂₃H₄₂O₃SiNa [M+Na⁺] 417.2801, found 417.2819.

Example 41

Macroketone 60: To a solution of TBS-macroketone 59 (194 mg, 0.492 mmol)in THF (15 mL) at rt was added HF•pyridine (3.5 mL). After stirring for15 h, the reaction mixture was carefully treated with MeOTMS (25 mL) andconcentrated under reduced pressure. Purification of the crude productby FC (hexane/EtOAc 10:1→4:1) afforded macroketone 60 (124 mg, 90%) as acolorless oil. [α]_(D) +77.6° (c 0.50, CHCl₃); IR (neat) 3566, 3022,3015, 2975, 2937, 2879, 1700, 1448, 1384, 1237, 1109, 1085, 979; ¹H-NMR(500 MHz, CDCl₃) δ 5.72 (ddd, J=15.0, 8.5, 6.0, 1H), 5.37 (dd, J=10.0,0.9, 1H), 5.31 (dd, J=15.6, 7.8, 1H), 3.47 (app t, J=8.5, 1H), 3.36 (dd,J=9.2, 1.2, 1H), 3.31 (s, 3H), 2.78 (br s, 1H), 2.51-2.45 (m, 2H),2.37-2.32 (m, 2H), 2.26-2.16 (m, 5H), 1.69 (d, J=1.3, 3H), 1.68-1.59 (m,2H), 1.55-1.50 (m, 2H), 0.95 (d, J=6.8, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ212.10, 135.23, 132.91, 130.26, 129.22, 83.69, 77.62, 56.45, 42.08,40.67, 32.57, 30.33, 28.57, 27.01, 23.22, 23.14, 12.61; MS (ESI) 303[M+Na⁺]; HRMS (FAB) calcd. for C₁₇H₂₈O₃Na [M+Na⁺] 303.1936, found303.1938.

Example 42

Secondary Alcohols 61 and 62: To a solution of alcohol 26 (360 mg, 1.15mmol) in CH₂Cl₂ (5 mL) at rt was added Dess-Martin periodinane (970 mg,2.29 mmol). After stirring for 1 h, the reaction mixture was treatedwith saturated aqueous Na₂S₂O₃ solution and saturated aqueous NaHCO₃solution. The organic layer was separated and the aqueous layer wasextracted with CH₂Cl₂ (3×). The combined organic layers were dried(Na₂SO₄) and concentrated under reduced pressure to afford thecorresponding aldehyde 27. Crude product 27 was dissolved in Et₂O (12mL) and i-PrMgCl (2.90 mL, 5.80 mmol, 2M in THF) was added at −78° C.After stirring for 5 h, the reaction mixture was treated with saturatedaqueous NH₄Cl solution. The organic layer was separated and the aqueouslayer was extracted with EtOAc (3×). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure. Purification ofthe crude product by FC (toluene/EtOAc 19:1) afforded (S)-secondaryalcohol 61 (186 mg, 50%) and (R)-secondary alcohol 62 (134 mg, 36%) ascolorless oils.

(S)-Secondary Alcohol 61: IR (neat) 3476, 2956, 2929, 2884, 2857, 1471,1462, 1378, 1251, 1127, 1096, 1080, 1032, 1006; ¹H-NMR (500 MHz, CDCl₃)δ 5.78-5.68 (m, 1H), 5.31-5.23 (m, 3H), 3.98 (d, J=9.8, 1H), 3.51-3.48(m, 2H), 3.24 (s, 3H), 2.73-2.68 (m, 1H), 1.78-1.69 (m, 1H), 1.66 (s,3H), 1.60 (br s, 1H), 1.03 (d, J=6.4, 3H), 0.91 (s, 9H), 0.89 (d, J=6.0,3H), 0.73 (d, J=6.9, 3H), 0.07 (s, 3H), 0.05 (s, 3H); ¹³C-NMR (125 MHz,CDCl₃) δ 135.37, 134.58, 133.16, 118.24, 85.74, 77.85, 75.73, 56.23,33.64, 30.96, 26.13, 19.51, 18.93, 18.48, 17.56, 15.66, −3.86, −4.57; MS(ESI) 379 [M+Na⁺]; HRMS (FAB) calcd. for C₂₀H₄₀O₃SiNa [M+Na⁺] 379.2644,found 379.2663.

(R)-Secondary Alcohol 62: IR (neat) 3378, 2955, 2931, 2919, 2872, 1466,1455, 1378, 1249, 1119, 1096, 1079, 1026; ¹H-NMR (500 MHz, CDCl₃) δ 5.59(ddd, J=17.3, 10.3, 8.2, 1H), 5.40 (dd, J=10.3, 1.4, 1H), 5.31 (dd,J=10.2, 1.5, 1H), 5.27 (dd, J=17.3, 1.4, 1H), 3.97 (d, J=9.2, 1H), 3.43(dd, J=7.4, 2.2, 1H), 3.37 (app t, J=8.1, 1H), 3.21 (s, 3H), 2.67-2.62(m, 1H), 1.80-1.73 (m, 1H), 1.67 (d, J=1.5, 3H), 1.37 (br s, 1H), 1.04(d, J=6.5, 3H), 0.91 (s, 9H), 0.90 (d, J=6.6, 3H), 0.76 (d, J=6.6, 3H),0.05 (s, 3H), 0.02 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 135.02, 134.13,133.88, 118.86, 86.39, 78.35, 75.92, 56.06, 33.16, 31.52, 26.15, 19.52,19.34, 18.59, 17.68, 13.74, −3.80, −4.84; MS (ESI) 379 [M+Na⁺]; HRMS(FAB) calcd. for C₂₀H₄₀O₃SiNa [M+Na⁺] 379.2644, found 379.2643.

Example 43

(S)-Isopropyl Ester 63: To a solution of alcohol 61 (55 mg, 0.154 mmol)in toluene (0.4 mL) at rt was added pyridine (62 μL, 0.772 mmol) and themixed anhydride of 6-heptenoic acid (The preparation of the mixedanhydride of 6-heptenoic acid and 2,4,6-trichlorobenzoyl chloride wasperformed exactly as for the mixed anhydride of 2,6-heptadienoic acidand 2,4,6-trichlorobenzoyl chloride (see above)) and2,4,6-trichlorobenzoyl chloride (1.5 mL, 0.75 mmol, 0.50M in toluene).After stirring 15 h, the reaction mixture was directly loaded onto asilica column and purified by FC (toluene) to afford (S)-isopropyl ester63 (54 mg, 75%) as a colorless oil. IR (neat) 2928, 2830, 1732, 1470,1378, 1247, 1125, 1096, 1032; ¹H-NMR (500 MHz, CDCl₃) δ 5.81-5.76 (m,2H), 5.41 (d, J=10.4, 1H), 5.31 (dd, J=10.3, 2.0, 1H), 5.24 (app d,J=17.2, 1H), 5.17 (app d, J=9.9, 1H), 5.00 (app d, J=17.1, 1H), 4.94 (d,J=5.8, 1H), 3.59 (dd, J=8.0, 1.8, 1H), 3.32 (app t, J=8.4, 1H), 3.20 (s,3H), 2.73-2.68 (m, 1H), 2.28 (app t, J=7.5, 2H), 2.08-2.04 (m, 2H),1.91-1.88 (m, 1H), 1.66-1.59 (m, 3H), 1.61 (s, 3H), 1.44-1.41 (m, 1H),0.91 (d, J=8.3, 3H), 0.90 (s, 9H), 0.84 (d, J=6.7, 3H), 0.77 (d, J=6.9,3H), 0.04 (s, 3H), −0.02 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 172.48,138.46, 135.43, 135.32, 129.62, 118.71, 114.62, 86.92, 78.07, 77.52,77.34, 55.83, 34.42, 33.39, 33.27, 29.71, 28.38, 26.22, 24.66, 19.22,18.53, 18.36, 12.69, −3.80, −4.96; MS (ESI) 489 [M+Na⁺]; HRMS (FAB)calcd. for C₂₇H₅₀O₄SiNa [M+Na⁺] 489.3376, found 489.3362.

Example 44

(R)-Isopropyl Ester 66: Preparation performed exactly as for(S)-isopropyl ester 63, affording (R)-isopropyl ester 66 in 70% yield.IR (neat) 2956, 2928, 2856, 1732, 1469, 1462, 1370, 1249, 1129, 1032;¹H-NMR (500 MHz, CDCl₃) δ 5.81-5.74 (m, 1H), 5.61 (ddd, J=17.6, 10.6,7.6, 1H), 5.45 (d, J=9.4, 1H), 5.33 (dd, J=10.4, 1.9, 1H), 5.29 (dd,J=17.0, 1.9, 1H), 5.15 (app d, J=9.8, 1H), 4.99 (dd, J=17.0, 1.9, 1H),4.95-4.93 (m, 1H), 3.40-3.38 (m, 2H), 3.21 (s, 3H), 2.82-2.76 (m, 1H),2.29 (app t, J=7.5, 2H), 2.08-2.03 (m, 2H), 1.97-1.90 (m, 2H), 1.64-1.61(m, 1H), 1.61 (d, J=1.3, 3H), 1.43-1.36 (m, 2H), 0.92 (d, J=6.6, 3H),0.91 (s, 9H), 0.89 (d, J=6.6, 3H), 0.79 (d, J=6.0, 3H), 0.05 (s, 3H),0.01 (s, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 172.82, 138.47, 135.59, 134.68,129.61, 118.97, 114.63, 86.41, 78.32, 77.72, 56.06, 34.42, 33.65, 33.38,29.71, 28.35, 26.18, 24.58, 19.38, 18.96, 18.61, 18.19, 12.89, −3.76,−4.91; MS (ESI) 489 [M+Na⁺]; HRMS (FAB) calcd. for C₂₇H₅₀O₄SiNa [M+Na⁺]489.3376, found 489.3363.

Example 45

(S)-Isopropyl Macrolactone 65: To a solution of (S)-isopropyl ester 63(25 mg, 0.053 mmol) in refluxing toluene (100 mL) was added Grubbs-IIcatalyst 16 (9 mg, 0.0107 mmol). After stirring for 15 min, the reactionmixture was cooled to rt and filtered through a silica plug(hexane/EtOAc 1:3). After evaporation of the solvent, crude product 64was dissolved in THF (3 mL) and treated with HF•pyridine (0.75 mL) atrt. After stirring for 40 h, the reaction mixture was carefully treatedwith MeOTMS (6 mL) and concentrated under reduced pressure. Purificationof the crude product by FC(CH₂Cl₂/EtOAc 9:1) afforded (S)-isopropylmacrolactone 65 (12 mg, 65%) as a colorless oil. [α]_(D) +25.1° (c 0.32,CHCl₃); IR (neat) 3479, 2967, 2926, 2876, 1724, 1448, 1373, 1257, 1237,1091; ¹H-NMR (500 MHz, CDCl₃) δ 5.70 (ddd, J=15.4, 8.5, 5.3, 1H), 5.33(dd, J=10.0, 0.9, 1H), 5.30 (d, J=7.0, 1H), 5.19-5.13 (m, 1H), 3.40-3.30(m, 2H), 3.28 (s, 3H), 2.99-2.95 (m, 1H), 2.76 (br s, 1H), 2.36-2.24 (m,2H), 2.20-2.08 (m, 2H), 1.99 (app dt, J=7.0, 6.9, 1H), 1.69 (d, J=1.3,3H), 1.62-1.52 (m, 4H), 0.94 (d, J=7.0, 3H), 0.91 (d, J=6.6, 3H), 0.86(d, J=6.9, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 172.97, 135.94, 133.83,130.09, 127.75, 86.47, 78.70, 55.98, 33.99, 32.80, 30.38, 29.82, 27.34,22.57, 21.38, 19.09, 18.05, 15.20; MS (ESI) 347 [M+Na⁺]; HRMS (FAB)calcd. for C₁₉H₃₂O₄Na [M+Na⁺] 347.2198, found 347.2187.

Example 46

(R)-Isopropyl Macrolactone 68: Preparation performed exactly as for(S)-isopropyl macrolactone 65, affording (R)-isopropyl macrolactone 68in 66% yield. [α]_(D) +21.3° (c 0.09, CHCl₃); IR (neat) 3499, 2967,2926, 2866, 1729, 1453, 1383, 1257, 1111; ¹H-NMR (500 MHz, CDCl₃) δ 5.65(app dt, J=15.5, 7.5, 1H), 5.58 (dd, J=10.7, 1.3, 1H), 5.35 (dd, J=15.5,6.0, 1H), 4.87 (d, J=7.6, 1H), 3.49 (dd, J=9.1, 6.0, 1H), 3.34 (s, 3H),3.27 (br d, J=8.8, 1H), 3.13-3.07 (m, 1H), 2.86 (br s, 1H), 2.34-2.15(m, 4H), 2.06-1.99 (m, 1H), 1.76 (d, J=1.6, 3H), 1.75-1.58 (m, 3H),1.47-1.41 (m, 1H), 0.98 (d, J=7.0, 3H), 0.93 (d, J=6.7, 3H), 0.92 (d,J=6.7, 3H); ¹³C-NMR (125 MHz, CDCl₃) δ 172.50, 132.45, 132.08, 131.58,128.26, 82.45, 80.74, 77.44, 56.67, 33.00, 32.66, 31.76, 30.56, 25.57,24.91, 22.44, 19.02, 18.96, 13.20; MS (ESI) 347 [M+Na⁺]; HRMS (FAB)calcd. for C₁₉H₃₂O₄Na [M+Na⁺] 347.2198, found 347.2196.

Example 47

Macrocyclic Secondary Alcohol 69 (diastereomeric mixture): To a solutionof macroketone 60 (4 mg, 0.014 mmol) in MeOH (0.3 mL) at rt was addedNaBH₄ (2 mg, 0.042 mmol). After stirring for 5 min, the reaction mixturewas carefully treated with 1M HCl (1 mL) and stirring was continued foranother 20 min. Then the reaction mixture was diluted with EtOAc, theorganic layer was separated, and the aqueous layer was extracted withEtOAc (4×). The combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure to afford a diastereomeric mixtureof macrocyclic secondary alcohol 69 (4 mg, 95%) as a colorless oil. IR(neat) 3405, 2931, 2922, 2856, 1446, 1380, 1106, 1090; ¹H-NMR (500 MHz,CDCl₃) δ 5.66-5.59 (m, 2H), 5.32 (app t, J=8.4, 2H), 5.27-5.19 (m, 2H),3.83-3.72 (m, 2H), 3.49 (s, 1H), 3.46-3.40 (m, 2H), 3.36 (app t, J=10.0,1H), 3.30 (s, 6H), 2.74 (br s, 2H), 2.59-2.46 (m, 2H), 2.31-2.26 (m,2H), 2.19-2.06 (m, 2H), 2.02-1.90 (m, 2H), 1.83-1.72 (m, 4H), 1.70 (s,6H), 1.68-1.13 (m, 12H), 0.94 (app t, J=6.3, 6H); ¹³C-NMR (125 MHz,CDCl₃) δ 136.43, 136.22, 134.53, 134.21, 129.52, 129.40, 129.28, 129.19,84.38, 84.14, 77.51, 77.42, 71.17, 70.66, 56.28, 56.22, 33.36, 33.30,32.87, 32.50, 32.21, 32.16, 30.47, 30.34, 26.93, 26.91, 26.83, 25.50,23.46, 23.37, 21.90, 19.67, 12.58, 12.44; MS (ESI) 305 [M+Na⁺]; HRMS(FAB) calcd. for C₁₇H₃₀O₃Na [M+Na⁺] 305.2093, found 305.2103.

Example 48

Macrocyclic Tertiary Alcohol 70 (diastereomeric mixture): To a solutionof macroketone 60 (5.5 mg, 0.020 mmol) in THF (0.4 mL) at 0° C. wasadded MeMgBr (66 μL, 0.200 mmol, 3M in Et₂O). After stirring for 5 min,the reaction mixture was treated with saturated aqueous NH₄Cl solutionand diluted with EtOAc. The organic layer was separated and the aqueouslayer was extracted with EtOAc (4×). The combined organic layers weredried (MgSO₄) and concentrated under reduced pressure to afford adiastereomeric mixture of macrocyclic tertiary alcohol 70 (6.0 mg, 95%)as a colorless oil. IR (neat) 3434, 2933, 2856, 1460, 1448, 1117, 1083;¹H-NMR (500 MHz, CDCl₃) δ 5.66-5.60 (m, 2H), 5.34-5.31 (m, 2H),5.24-5.17 (m, 2H), 3.46-3.32 (m, 6H), 3.30 (s, 6H), 2.80-2.70 (m, 2H),2.61-2.51 (m, 2H), 2.30-2.26 (m, 2H), 2.17 (br s, 1H), 2.14-2.01 (m,2H), 1.95-1.82 (m, 2H), 1.77-1.60 (m, 2H), 1.70 (s, 6H), 1.58-1.36 (m,8H), 1.34-1.14 (m, 5H), 1.20 (s, 6H), 1.02 (app t, J=7.2, 2H), 0.94-0.92(m, 6H); ¹³C-NMR (125 MHz, CDCl₃) δ 136.55, 136.44, 134.45, 134.35,129.44, 129.32, 84.34, 84.24, 72.90, 72.80, 56.27, 56.23, 38.71, 38.60,38.48, 38.43, 32.10, 32.09, 30.92, 30.56, 29.69, 29.32, 29.24, 27.41,27.37, 26.79, 24.27, 23.34, 23.33, 21.91, 21.14, 12.64, 12.57; MS (ESI)319 [M+Na⁺]; HRMS (FAB) calcd. for C₁₈H₃₂O₃Na [M+Na⁺] 319.2249, found319.2264.

Example 49

Macrocyclic CF₃-Alcohol 71 (major): To a solution of macroketone 60 (10mg, 0.036 mmol) and TMSCF₃ (27 μL, 0.180 mmol) in THF (0.6 mL) at rt wasadded a catalytic amount of TBAF. After stirring for 1 h, the reactionmixture was treated with excess TBAF and stirred for another 5 h. Thereaction mixture was concentrated under reduced pressure. Purificationof the crude product by FC (hexane/EtOAc 3:1) afforded a diastereomericmixture of alcohol 71 (10 mg, 80%) as a colorless oil. Furtherpurification by FC (hexane/EtOAc 7:1→3:1) provided the major isomer 71in pure form as a colorless oil. IR (neat) 3409, 2963, 2931, 2922, 1457,1244, 1150, 1112; ¹H-NMR (500 MHz, CDCl₃) δ 5.64 (ddd, J=17.2, 9.4, 5.1,1H), 5.34 (d, J=10.7, 1H), 5.19 (dd, J=17.2, 8.2, 1H), 3.43 (app t,J=9.0, 1H), 3.36 (app d, J=9.5, 1H), 3.30 (s, 3H), 2.87 (br s, 1H),2.56-2.47 (m, 1H), 2.31-2.26 (m, 1H), 2.11-2.03 (m, 1H), 2.00-1.84 (m,2H), 1.71-1.68 (m, 2H), 1.69 (s, 3H), 1.69-1.38 (m, 4H), 1.30-1.22 (m,2H), 0.99 (app t, J=7.3, 1H), 0.93 (d, J=7.0, 3H); ¹³C-NMR (125 MHz,CDCl₃) δ 136.38, 133.31, 129.78, 129.45, 83.77, 56.33, 32.00, 31.02,30.41, 29.72, 26.99, 25.09, 23.93, 23.27, 20.20, 19.63, 12.74; MS (ESI)373 [M+Na⁺]; HRMS (FAB) calcd. for C₁₈H₂₉F₃O₃Na [M+Na⁺] 373.1966, found373.1971.

Example 50

Macrooxime 72 (diastereomeric mixture): A solution of macroketone 60 (5mg, 0.018 mmol) and NH₂OH.HCl (12 mg, 0.178 mmol) in pyridine (0.3 mL)was heated to 45° C. for 3 h. The reaction mixture was concentratedunder reduced pressure and the crude product was purified by FC(hexane/EtOAc 1:1) to afford a diastereomeric mixture of macrooxime 72(4 mg, 70%) as a colorless oil. IR (neat) 3326, 2930, 1447, 1109, 1086,981; ¹H-NMR (500 MHz, CDCl₃) δ 5.72-5.64 (m, 2H), 5.37 (d, J=9.1, 2H),5.31-5.25 (m, 2H), 3.50-3.45 (m, 2H), 3.38-3.35 (m, 2H), 3.32 (s, 6H),2.82 (br s, 2H), 2.62-2.57 (m, 2H), 2.43-2.36 (m, 2H), 2.29-2.04 (m,14H), 1.76 (d, J=1.6, 3H), 1.71 (d, J=1.8, 3H), 1.56-1.48 (m, 6H),1.27-1.24 (m, 2H), 0.97 (d, J=6.8, 3H), 0.96 (d, J=6.8, 3H); ¹³C-NMR(125 MHz, CDCl₃) δ 161.91, 161.66, 135.46, 135.30, 134.03, 133.76,129.76, 129.64, 129.29, 129.19, 83.93, 83.89, 77.65, 77.48, 56.42,33.66, 32.57, 32.51, 32.42, 30.62, 30.41, 30.29, 28.22, 27.01, 26.94,26.70, 26.64, 24.42, 23.51, 23.13, 12.67; MS (ESI) 318 [M+Na⁺]; HRMS(FAB) calcd. for C₁₇H₂₉NO₃Na [M+Na⁺] 318.2045, found 318.2049.

Example 51

Biotinylated Macrohydrazone 73 (diastereomeric mixture): A solution ofmacroketone 60 (6 mg, 0.021 mmol) and biotin-dPEG₄-hydrazide (13 mg,0.026 mmol) in EtOH (0.3 mL) was heated to 55° C. for 1 h. The reactionmixture was concentrated under reduced pressure and the crude productwas purified by FC(CH₂Cl₂/MeOH 4:1) to afford a diastereomeric mixtureof biotinylated macrohydrazone 73 (12 mg, 75%) as a colorless oil. IR(neat) 3291, 2930, 2872, 1703, 1691, 1680, 1668, 1540, 1459, 1261, 1104;¹H-NMR (500 MHz, CDCl₃) δ 9.56 (s, 0.4H), 9.50 (s, 0.4H), 9.23 (s,0.6H), 9.10 (s, 0.6H), 6.77-6.73 (m, 2H), 6.58 (s, 0.6H), 6.50 (s, 6H),6.12 (s, 0.4H), 6.08 (s, 0.4H), 5.70-5.63 (m, 2H), 5.42-5.34 (m, 2H),5.32-5.25 (m, 2H), 5.18 (s, 0.8H), 5.04 (s, 1.2H), 4.50-4.47 (m, 2H),4.37-4.32 (m, 2H), 3.83-3.79 (m, 4H), 3.68-3.55 (m, 32H), 3.48-3.41 (m,6H), 3.39-3.28 (m, 8H), 3.16-3.14 (m, 2H), 2.97-2.89 (m, 5H), 2.82 (s,0.6H), 2.78 (s, 0.4H), 2.74-2.70 (m, 2H), 2.64-2.56 (m, 4H), 2.30-2.22(m, 10H), 2.20-2.02 (m, 4H), 2.17 (s, 3H), 1.77 (s, 3H), 1.75-1.64 (m,12H), 1.25-1.24 (m, 2H), 0.95 (d, J=7.0, 3.6H), 0.92 (d, J=6.6, 2.4H);¹³C-NMR (125 MHz, CDCl₃) δ 173.95, 173.74, 173.33, 173.22, 163.73,163.67, 163.49, 160.94, 160.65, 156.08, 155.65, 135.39, 135.02, 134.78,133.92, 133.65, 132.80, 132.62, 130.73, 130.61, 129.71, 129.56, 129.39,128.99, 128.92, 83.99, 83.83, 83.79, 83.70, 77.64, 77.53, 77.46, 77.41,70.42, 70.38, 70.14, 70.04, 69.92, 61.77, 61.73, 60.08, 56.46, 56.40,40.61, 40.54, 39.13, 39.10, 36.29, 35.86, 35.77, 33.17, 32.68, 32.44,30.92, 30.67, 30.46, 30.36, 29.68, 28.07, 27.41, 26.88, 26.81, 26.58,25.51, 25.45, 24.59, 23.63, 23.49, 23.26, 12.71, 12.66; MS (ESI) 768[M+H⁺]; HRMS (FAB) calcd. for C₃₈H₆₆N₅O₉S [M+H⁺] 768.4581, found768.4581.

Example 52 Preliminary Biological Data

1. Tube Formation Assay (Table 1):

A protocol was designed based on the instructions from the provider (BDBioscience, San Jose, Calif.). Briefly, wells of a 48 well culture dishwere covert with 150 μL matrigel and the matrigel was gelatinized for 30min at 37° C. A 80-90% confluent HUVEC (BD Bioscience, San Jose, Calif.)culture was trypsin treated, the detached cells were collected bycentrifugation and resuspended in EGM-2 media (BD Bioscience, San Jose,Calif.). Cell concentration was adjusted to 100.000 cells/mL. 400 μL ofthe cell suspension were filled in the matrigel coated wells, and asolution of the inhibitor was added to the intended final concentration.The plates were incubated at 37° C. with 5% CO₂ for 16-18 h. Media wasremoved, and the matrigel surface was washed twice with 500 μL PBSbefore cells were labeled with 250 μL 8 μM Calcein AM (Pierce, Rockford,Ill.) in PBS for 30 min at 37° C. After two additional washing steps(500 μL PBS) cells were visualized under an inverted microscope.Fluorescence was excited at 488 nm and recorded at 538 nm. The minimumeffect concentration was defined as the minimal inhibitor concentrationthat caused a definite disturbance of the complexity of the formed tubenetwork.

2. Wound Healing Assay (Table 2)

The wound healing assay was performed based on the method described byNakae et al. (Nakae et al., J. Antibiotics (2000), 53, 1130-1136).Briefly, adherent cells were grown in a suitable media to confluence(e.g. KYSE-520 cells in RPMI-1640 with 10% FBS). Cells were starved for24 h in serum free media. A scratch (ca 0.5 mm) was applied and the celllayer was washed twice with PBS after removal of the media. Fresh, serumfree media with the test compound at the desired concentration was addedand the cells were incubated for 28 to 30 h at 37° C., 5% CO₂. Thescratch size was compared to that observed for cells exposed to 100 μMMigrastatin. Test compounds associated with a scratch size equal to orlarger than that observed for cells exposed to 100 μM Migrastatin weredeemed to have cell migration inhibitory activity at least equal toMigrastatin.

3. Chamber Cell Migration Assay (Table 3)

Cells were grown in an appropriate media to 70 to 80% confluence andincubated in serum and growth factor free media for 24 h. Cells weredetached by trypsin treatment, collected by centrifugation andresuspended in serum free media to a final concentration of 150,000cells/mL. 400 μL of the cell suspension were loaded into a fibronectincoated insert for 24 well multidishes. 750 μL fully supplemented mediawere applied to the compartment under the insert. To both chambers theinhibitor was added at the intended concentration and the plates wereincubated for 36 h at 37° C., 5% CO₂. The media from both chambers wasaspirated, the lower section was filled with 300 μL CyQuant assaysolution (Molecular Probes, Eugene, Oreg.), and incubated at roomtemperature for 5 min. The resulting CyQuant assay solution wastransferred to the cavities of a 96 well microtiter plate and thefluorescence signal was recorded in an appropriate reader. The CyQuantdye forms a highly fluorescent complex with DNA, thus the fluorescencesignal is proportional to the number of cells that migrated through themembrane in the presence of the test compound (N^(inh)). A positivecontrol (i.e., without a test compound in the growth media) was carriedout according to the procedure described above, except that no testcompound was added. The positive control fluorescent reading correlateswith the number of cells that migrate through the membrane in theabsence of inhibitor (N⁺). A negative control (i.e., without a testcompound and without attractants (e.g., growth factors, serum) in thegrowth media) was carried out according to the procedure describedabove, except that no test compound and attractants were added. Thenegative control fluorescent reading correlates with the number of cellsthat migrate through the membrane through non-directed processes (N⁻).The anti-migratory effect of a test compound is determined by the ratio(N^(inh)−N⁻)/(N⁺−N⁻).

Example 53

Chamber Cell Migration Assay (Tables 4 and 6): Cell migrations wereassayed with Boyden chambers [8.0 μm pore size, polyethyleneterephthalate membrane, FALCON cell culture insert (Becton-Dickinson)].4T1 mouse breast tumor cells or HUVECs were trypsinized and counted. 300μl of 5-10×10⁴ cells in serum-free medium was added to the upper chamberand 500 μl of medium with 10% fetal bovine serum (FBS) was added to thelower chamber. The transwells were incubated for 6-8 h at 37° C. withdifferent concentrations of chemical compound in both upper and lowerchamber. Cells on the inside of the transwell inserts were removed witha cotton swab, and cells on the underside of the insert were fixed andstained. Photographs of three random regions were taken and the numberof cells was counted to calculate the average number of cells that havetransmigrated.

Examplary effects of migrastatin analogs, macrolactone 48 andmigrastatin 1, on 4T1 tumor cell migration are shown in FIG. 2.

Example 54

Mouse Plasma Stability Studies (Table 5): HPLC conditions: The sample isinjected and separated using an Inertsil ODS3 6u 3×150 mm column with amobile phase of MeCN and water (50% for migrastatin) at a flow of 0.4mL/min, monitored at 220 nm at 0.02 AUFS (the retention time formigrastatin is ca. 4 min, the identity of this peak was confirmed bymass spectral analysis). Incubation and sample preparation conditions: Asolution (ca. 30 mM) of chemical compound (Table 5) in DMSO is prepared.2 μL of the solution is added to a mixture containing 200 μL of mouseplasma and 800 μL of PBS. The resulting solution is put into a waterbath at 37° C., and 100 μL, of sample is withdrawn at 10, 20, 30, 45,and 60 min. The precipitate is removed by centrifugation and 20 μL ofthe supernatant is injected onto the HPLC.

Example 55

Cell Proliferation Assay: 4×10⁴ of 4T1 tumor cells in RPMI-1640 mediumcontaining 10% FBS were seeded into wells of 96-multiwell plates(Becton-Dickinson) in the presence or absence of chemical compounds andthen incubated at 37° C. for 48 h. An MTT kit (Cell Proliferation Kit I,Roche) (a colorimetric assay) was used to quantify cell proliferationand viability. The number of living cells, thus the total metabolicactivity, directly correlates to the amount of purple formazan crystalsformed (monitored by the absorbance).

Examplary effects of migrastatin 1, and migrastatin analogs,macrolactone 48, macrolactam 55, and macroketone 60 on 4T1 tumor cellproliferation are shown in FIG. 3.

Example 56

Inhibition of metastasis of mouse breast tumors by migrastatin analogsin mice. 4T1 mouse breast tumor cell line was isolated from a singlespontaneously arising mammary tumor from a BALB/BfC3H mouse (MMTV+).⁶²The 4T1 tumor closely mimics human breast cancer in its anatomical site,immunogenecity, growth characteristics, and metastatic properties.⁶³From the mammary gland, 4T1 tumor spontaneously metastasizes to avariety of target organs including the lung, bone, brain, and liverthrough primarily a hematogenous rout.⁶⁴

To assess the efficacy of therapeutic application of migrastatin analogsin the 4T1 murine mammary carcinoma models, we administered migrastatinanalogs (macroketone and macrolactam) to BALB/c mice carrying the 4T1tumors.

Female BALB/c mice (6-8 week old) were purchased from the JacksonLaboratory (Bar Harbor, Me.). All mice were housed at the Weill MedicalCollege of Cornell University Animal Facilities in accordance with thePrinciples of Animal Care (NIH publication no. 85-23, revised 1985). 4T1tumor cells (1×10⁵) were injected subcutaneously into the abdominalmammary gland area of mice in 0.1 ml of a single-cell suspension inphosphate buffered saline (PBS) on Day 0. The dosage of tumorimplantation was empirically determined to give rise to tumor of ˜10 mmin diameter in untreated wild type mice in 21-23 days. On Day 7, whenthe tumors averaged in size ˜4-5 mm in diameter, migrastatin analogs orcontrol PBS saline were given every day by intraperitoneal injection at10 mg/kg or 20 mg/kg per mouse until Day 25. On Day 28, the mice weresacrificed. This regiment of migrastatin analogs was well tolerated withno signs of overt toxicity. Every group included five mice. Primarytumors were measured using electronic calipers on the day when the micewere sacrificed. Tumor size was the square root of the product of twoperpendicular diameters. Numbers of metastatic 4T1 cells in lung weredetermined by the clonogenic assay.⁶³ In brief, lungs were removed fromeach mouse on Day 28, finely minced and digested in 5 ml of enzymecocktail containing 1×PBS and 1 mg/ml collagenase type IV for 2 hours at37° C. on a platform rocker. After incubation, samples were filteredthrough 70 uM nylon cell strainers and washed twice with PBS. Resultingcells were suspended and plated serially diluted in 10 cm tissue culturedishes in medium RPMI1640 containing 60 uM thioguanine for clonogenicgrowth. 6-Thioguanine-resistant tumor cells formed foci after 14 days,at which time they were fixed with methanol and stained with 0.03%methylene blue for counting.

Example 57

Treatment 4T1 tumor lung metastasis in syngeneic mice with migrastatinanalogs (See, FIG. 4). 4T1 tumor cells (10⁵) were injected s.c. in theabdominal mammary gland with 0.1 ml of a single-cell suspension.Macroketone or macrolactam at 10 mg/kg or 20 mg/kg was given i.p. on Day7 when the tumor size was about 5 mm in diameter, and every day untilDay 25. On Day 28, the mice were sacrificed. Each group comprised fivemice. Lung metastasis was measured by the 6-thiogunine clonogenic assay.The mean and standard deviation are presented. In the control group(daily PBS injection), there were 61300±18900 colonies. In the grouptreated with 10 mg/kg of macroketone, there were 3875±2525 colonies(˜94% inhibition of lung metastasis). In the group treated with 20 mg/kgof macroketone, there were 650±575 colonies (˜99% inhibition of lungmetastasis). In the group treated with 10 mg/kg of macrolactam, therewere 5333±1778 colonies (˜91% inhibition of lung metastasis). In thegroup treated with 20 mg/kg of macrolactam, there were 5675±6263colonies (˜91% inhibition of lung metastasis).

Example 58

Effect of migrastatin analogs on 4T1 tumor cell growth (See, FIG. 5).4T1 tumor cells (10⁵) were injected s.c. in the abdominal mammary glandwith 0.1 ml of a single-cell suspension. Macroketone or macrolactam at10 mg/kg or 20 mg/kg was given i.p. on Day 7 when the tumor size wasabout 5 mm in diameter, and every day until Day 25. On Day 28, primarytumors were measured using electronic calipers. Treatment with themigrastatin analogs did not slow the growth of 4T1 tumors significantlycompared to the control PBS saline. We noticed that macroketone at 10mg/kg had a minor effect on tumor growth in mice since the final tumorsize was a little smaller. We dissolved all compounds in DMSO and thendiluted into PBS. The final concentration of DMSO was 1% in all cases.The control mice were injected with 1% DMSO in PBS. Each group wascomprised of five mice. The mean and standard deviation are presented.

Example 59

Wound-Healing Assay (See FIG. 6). 4T1 mouse breast tumor cells inRPMI-1640 medium containing 10% fetal bovine serum (FBS) were seededinto wells of 24-multiwell plates (Becton-Dickinson). After cells grewto confluence, wounds were made with sterile pipette tips. Cells werewashed with Phosphate Buffered Saline (PBS) and refreshed with growthmedium containing different concentrations of chemical compounds. Afterovernight incubation at 37° C., cells were fixed and photographed.

REFERENCES

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1. A method for treating breast tumor metastasis in a subjectcomprising: administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising a compound having one ofthe following structures:

or pharmaceutically acceptable salt thereof; wherein R₁ and R₂ arehydrogen or lower alkyl; R₃, R₅ and R₆ are C₁₋₆ alkyl; the bond

 is a single bond or a double bond; R₄ is halogen, —OR^(4A),—OC(═O)R^(4A) or —NR^(4A)R^(4B); wherein R^(4A) and R^(4B) areindependently hydrogen; a nitrogen protecting group selected from acarbamate, an amide, a cyclic imide derivative, an N-alkyl amine, anN-aryl amine, an imine derivative or an enamine derivative or an oxygenprotecting group selected from a substituted methyl ether, a substitutedethyl ether, a substituted benzyl ether, a silyl ether, an ester, acarbonate, a cyclic acetal or a ketal; or R^(4A) and R^(4B), takentogether with the nitrogen atom to which they are attached, form a C₃₋₂₀heterocyclic or C₃₋₁₄ heteroaryl moiety; or R₄, taken together with thecarbon atom to which it is attached forms a moiety having the structure

or R^(4A) and R^(4B) are independently a C₁₋₁₆ alkyl group optionallysubstituted with one or more of C₁₋₂₀ aliphatic; C₃₋₁₄ aryl; C₃₋₁₄heteroaryl; C₁₋₂₀ alkylC₃₋₁₄aryl; C₁₋₂₀ alkylC₃₋₁₄heteroaryl, C₃₋₁₄aryloxy; C₁₋₂₀ heteroalkoxy, C₃₋₁₄ heteroaryloxy; C₁₋₂₀ alkylthio; C₃₋₁₄arylthio; heteroC₁₋₂₀alkylthio; heteroC₃₋₁₄arylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₁R_(x); —OCON(R_(x))₂; —N(R_(x))₂; S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) is independently C₁₋₂₀ aliphatic,heteroC₁₋₂₀aliphatic, C₃₋₁₄ aryl, C₃₋₁₄ heteroaryl, C₁₋₂₀ alkylC₃₋₁₄arylor C₁₋₂₀ alkylC₃₋₁₄heteroaryl; X₁ is O, S, NR^(X1) or CR^(X1)R^(X2);wherein R^(X1) and R^(X2) are independently hydrogen, halogen, or asubstituted or unsubstituted C₁₋₂₀ alkyl, heteroC₁₋₂₀alkyl,cycloC₃₋₁₀alkyl, heterocyclo C₃₋₁₀alkyl, C₃₋₁₄ aryl or C₃₋₁₄ heteroaryl,or a nitrogen protecting group selected from a carbamate, an amide, acyclic imide derivative, an N-alkyl amine, an N-aryl amine, an iminederivative or an enamine derivative; Q is hydrogen, halogen, —CN,—S(O)₁₋₂R^(Q1), —NO₂, —COR^(Q1), —CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2),—NR^(Q1)C(═O)OR^(Q2), —CONR^(Q1)R^(Q2), or a substituted orunsubstituted C₁₋₂₀ aliphatic, heteroC₁₋₂₀aliphatic, C₃₋₂₀ alicyclic,heteroC₃₋₂₀alicyclic, C₃₋₁₄ aryl or C₃₋₁₄ heteroaryl moiety, or—WR^(Q1); wherein W is independently O, S or NR^(Q3) and each occurrenceof R^(Q1), R^(Q2) and R^(Q3) is independently hydrogen, or a substitutedor unsubstituted C₁₋₂₀ aliphatic, heteroC₁₋₂₀aliphatic, C₃₋₂₀ alicyclic,heteroC₃₋₂₀ alicyclic, C₃₋₁₄aryl or C₃₋₁₄ heteroaryl moiety; Y₂ ishydrogen, or a substituted or unsubstituted C₁₋₂₀ alkyl,heteroC₁₋₂₀alkyl, cyclo C₃₋₁₀alkyl, heterocycloC₃₋₁₀alkyl, C₃₋₁₄aryl, orC₃₋₁₄ heteroaryl moiety; or —WR^(Y1); W is O or NH; and R^(Y1) andR^(Y2) are independently hydrogen, or a substituted or unsubstitutedC₁₋₂₀ aliphatic, heteroC₁₋₂₀aliphatic, C₃₋₂₀ alicyclic,heteroC₃₋₂₀alicyclic, C₃₋₁₄ aryl or C₃₋₁₄ heteroaryl moiety; wherein forthe compound of formula (a), when X¹ is O and the bond

 is a double bond, Q is hydrogen, halogen, —CN, —S(O)₁₋₂R^(Q1), —NO₂,—COR^(Q1), —CO₂R^(Q1), —NR^(Q1)C(═O)R^(Q2), —NR^(Q1)C(═O)OR^(Q2),—CONR^(Q1)R^(Q2), or —WR^(Q1); wherein W is independently O, S orNR^(Q3) and each occurrence of R^(Q1), R^(Q2) and R^(Q3) isindependently hydrogen, or a substituted or unsubstituted C₁₋₂₀aliphatic, heteroC₁₋₂₀aliphatic, C₃₋₂₀ alicyclic, heteroC₃₋₂₀ alicyclic,C₃₋₁₄aryl or C₃₋₁₄ heteroaryl moiety; wherein the composition isadministered at a dose corresponding to a dose in a mouse that isbetween about 10 mg/kg and about 20 mg/kg.
 2. The method of claim 1wherein in the composition, the compound has one of the followingstructures:


3. The method of claim 1, further comprising administering a cytotoxicagent.
 4. The method of claim 3, wherein the cytotoxic agent is ananticancer agent.
 5. The method of claim 1, further comprisingadministering a palliative agent.
 6. The method of claim 1, wherein inthe composition the compound has one of the following structures:

or pharmaceutically acceptable salt thereof.
 7. The method of claim 6,wherein in the compound of the composition R₁ and R₂ are each hydrogen.8. The method of claim 6, wherein in the compound of the composition R₅and R₆ are each methyl.
 9. The method of claim 6, wherein in thecompound of the composition R₃ is lower alkyl.
 10. The method of claim9, wherein in the compound of the composition R₃ is methyl.
 11. Themethod of claim 6, wherein in the compound of the composition R₄ is OH.12. The method of claim 6, wherein in the compound of the composition Y₂is lower alkyl and R^(Y1) is hydrogen or lower alkyl.
 13. The method ofclaim 6, wherein in the compound of the composition R^(Y1) is hydrogenand Y₂ is CF₃.
 14. The method of claim 6, wherein in the composition thecompound has the structure:

or a pharmaceutically acceptable salt thereof, wherein n is 3; Y₂ islower alkyl substituted with one or more halogen atoms selected from F,Cl, Br and I, and R^(Y1) is hydrogen or lower alkyl.
 15. The method ofclaim 14, wherein R^(Y1) is hydrogen and Y₂ is CF₃.
 16. The method ofclaim 6, wherein in the composition the compound has one of thefollowing structures:

or a pharmaceutically acceptable salt thereof, wherein Y₂ is lower alkylsubstituted with one or more halogen atoms selected from F, Cl, Br andI, and R^(Y1) is hydrogen or lower alkyl.
 17. The method of claim 16,wherein R^(Y1) is hydrogen and Y₂ is CF₃.
 18. A method for treatingbreast tumor metastasis in a subject comprising: administering to asubject in need thereof a therapeutically effective amount of acomposition comprising a compound having the following structure:

or a pharmaceutically acceptable salt thereof; wherein X₁ is CH₂, NH orO; Y₁ and Y₂ are independently OH, C(R^(Y))₃ or Y₁ and Y₂ taken togetherwith the carbon atom to which they are attached are —C═O, wherein R^(Y)is halo; the bond

is a single bond or a double bond; R₆ is H or lower alkyl; R₅ is H orlower alkyl; R₄ is OH; and R₃ is lower alkyl; wherein the composition isadministered at a dose corresponding to a dose in a mouse that isbetween about 10 mg/kg and about 20 mg/kg.